Johnny Test concerns the adventures of Johnny Test, an average 11-year-old boy, who lives with his genius twin sisters, Susan and Mary, and his talking dog, Dukey, in the town of Porkbelly. As a recurring gag, they use their young brother as their test subject for various experiments with which they try to impress Gil, their next-door neighbor. Sometimes, Johnny would also be given his sisters gadgets or superpowers to use in combat or other situations. At times Johnny will borrow some of the devices and special powers that the twin sisters had developed and invented to do their own dirty work, and then try to undo disasters and messes that they have created, as well as to fight evil villains found in Porkbelly.
Johnny's dad gets fed up with his filthy bathroom and demands Johnny to clean it up but when Johnny uses his sisters' special germ foam and his dad's cleaning products, problems arise as it causes the germs to grow out of control.
Johnny heads to summer camp and battles in a boating competition against rival rich-kid campers.
Johnny invites everyone to his house for a snowball fight, but forgets to put the word "fight" on the invitation, which misleads everyone into believing it's actually a "Snow Ball" dance.
The Tests try to get Johnny a new toy when it sells out in the local shopping mall.
Susan and Mary turn Johnny and Dukey into girls so that they can win the grand prize at a roller derby match, but things quickly get difficult when a blow on the head gives Dukey amnesia, and he thinks that he is a real derby queen.