In Rock University Presents the Naked Brothers Band, the player takes on the roles of the members of The Naked Brothers Band as it goes on tour around the country, with the proceeds going to charity. However, the older brother of the tour promoter is trying to steal the show by following the band around the country and putting up opposing bands to play at the same time, attempting to disgrace the band and earn the money for themselves.
In each band competition, actual Naked Brothers Band songs play as the player takes turns between each of the members of the band and "play" their instrument for the segment. Each instrument (including vocals) has its own gameplay mechanic, all rhythm based. The more successful hits the player gets in a row, the larger the multiplier, and the better show the band puts on, with fancy maneuvers by the band members and more visually entertaining stage and environment. At the end of the show, the player can optionally save the performance for replay in the future. A "Music 101" tutorial mode allows the player to practice each instrument before he or she ever steps foot on the stage - or later on in the game if the player is in need a refresher.
As the tours proceeds, the player can unlock additional wardrobes for all members of the band, and mix and match between each member.
There are some additional features to the game beyond the tour. In "Composer" mode, the player can create their own rhythms to his or her favourite songs, which can be shared with friends via WiFi. In "Video" mode, the player can modify recordings of previous performances, selecting predefined camera angles, adding special effects, etc. It is also possible to view past cut-scenes up to the point you have progressed to.
Head-to-head battles are also possible over Wifi, with the best score winning the match.