The game of Powerball has been one of the signature events of American Gladiators in both runs of the series.

The two contenders competed in a 45 second game against three Gladiators. Each contender had their own colored scoring balls which were in two separate bins, and had to try to place them into one of five scoring cylinders while avoiding the Gladiators. The rims of the scoring cylinders were sized such that the contenders generally had to slam dunk the ball into the cylinders to score; a ball thrown from a distance and getting into a scoring cylinder was a legal goal, but scoring in this manner was very difficult and thus rarely occurred. A contender was required to forfeit the ball in hand and pick up a new ball if, before scoring a goal, he or she was tackled to the floor or out of bounds by a Gladiator, or dropped the ball onto the floor.

In the first half of season one, the event was conducted on a half-circle shaped field, with two buckets (one for each player) in the center and the scoring cylinders on the edge of the half-circle. 15 points were awarded for each score in this format. From the second half of season one onward, the event was conducted on a larger, rectangular playing field with two ball buckets, each in a semicircular safe zone on either end of the field. Contenders were required to alternate which end of the field they took their scoring balls from. The scoring cylinders were spread out more, and one was placed in the center of the playing field, with goals scored there worth more points. This change was made for the game to more resemble football and because it allowed for more camera angles to be shown. For the remainder of season one, outer cylinder goals were worth one point and center cylinder goals were worth two. After that, the amount of points varied from 1-3 for outer cylinder goals and 2-5 for center cylinder goals, depending on the season and the round of play.

For the first season the scoring cylinders were basic stand-up cylinders; for the following seasons a design with a rounded bottom was used (perhaps to prevent the cylinders from falling down or breaking, the latter of which happened several times during season one when the weight of contender and Gladiator crashing onto it was enough to break the plastic the cylinder was made of).

One standing Powerball rule was that the Gladiators could not be excessively rough with the contenders (tackling them hard, hitting above the shoulders, etc.). Violators of this rule were immediately removed from the game, and the event would resume with the remaining Gladiators. In addition, a warning would be issued should the Gladiator move into one of the contender safe zones. During the second half of Season 1, all tackling was prohibited by Gladiators. Tackling was defined as wrapping up the contender and taking them down, pushing a contender down was legal. Initially, this was penalized by awarding a score to the contender, but later in the season, Gladiators were disqualified for tackling. This led to a round where two Gladiators were disqualified and only a single Gladiator was left to defend against both contenders. However, tackling was later legalized because it was seen that the game did not have enough contact and action without it. Gladiators could still be disqualified for excessively rough tackles.

A variant of this game, Super Powerball, was played in Season 4 only. Played only as the seventh game (Crunch Time), it featured three cylinders placed in a line in the middle of the Powerball field and two Gladiators. Three points were given for outer cylinder goals, and five for a center cylinder goal.

For the 2008 revival, players were given 60 seconds against the Gladiators, with the 4 outer cylinders worth 2 points and the center cylinder worth 3 points. The rims of the scoring cylinders were also widened considerably, thus making it easier for contenders to throw the balls into the cylinders from a distance, and forcing the Gladiators to not only stop the contender, but also to prevent the contender from being able to shoot the ball into the goal (although the Gladiators are allowed to deflect such shots as well). The sidelines of the playing field were removed and replaced with a padded retaining wall, removing the Gladiators' "out of bounds" option and requiring them to either tackle the contenders to the floor or strip the ball from their hands. For the second season of the revival, the rims of the scoring cylinders were downsized to prevent balls from being thrown in from a distance, and the points were reduced from 2/3 points to 1/2 points.