Sam and Max - a canine shamus and a hyperkinetic rabbit - are the freelance police: private investigators who receive missions from and are answerable to "The Commissioner". Upon the completion of a rescue mission from the clutches of a mad scientist, the duo drives through the opening credits and immediately receives a new assignment: Bruno the Bigfoot is missing from the circus and seems to have kidnapped Trixie, the Giraffe-Neck Girl. Sam and Max are on the case and will follow the mystery all across the United States, even if it takes them to such locations as The World's Largest Ball of Twine and The Mount Rushmore Dinosaur Tarpit.

Sam & Max Hit the Road is a puzzle-solving point-and-click adventure game. The player directly controls Sam, though Max will usually follow closely behind and is even available to use directly as an item in the inventory. Sam is able to be directed anywhere on the screen, look at objects, try to use objects, try to speak to objects and use items from his inventory on objects. Sam and Max must unravel the mystery piece by piece in order to unlock new locations on their map. They can travel between these locations at any time by using the car keys on their police car.

During conversation, the player is able to make Sam ask questions of persons, speak non-sequitur exclamations, or ask about specific objects, people, and locations (which are also unlocked as clues are discovered). The verb interface from previous LucasArts adventure games has been modified to icon-based commands, allowing the entire screen to be used for the playing area, similarly to Sierra titles.

The game also features a variety of mini-games, which can be accessed at any time once found. These include Highway-Surfing: a game where the player must jump over exit signs on the interstate, Car Bomb: a variant of Battleship, a Sam & Max coloring book, a Sam & Max dress-up game, and a Whac-a-Mole variant entitled Wak-A-Rat. The CD version of the game includes voice-overs for all the conversations.