The heist may only be initiated when a player is operating as a CEO or VIP Organization or a Motorcycle Club, with the first time playthrough focused on the protagonist conducting the heist to secure documents for Miguel Madrazo, the son of Martin Madrazo, being held by Strickler. In addition to this, players can also steal additional valuables from secure storage sites, which increase the overall payout of the heist. This includes going in aggressive, using stealth to neutralize hostiles, or conducting subterfuge by using disguises to get around guards.

The focus of the heist is to secure a major target being held in a vault beneath Strickler's secure compound and escape with it, using any methods to reach the island and breach into the vault. The Cayo Perico Heist focuses on players conducting a major heist against Juan Strickler, a notorious drug lord known to many as El Rubio, who maintains an island compound in the Caribbean which houses a collection of valuables, is home to a major part of his drug operations, and maintains tight security measures and heavily armed guards, including gunboats, an attack helicopter, and surface-to-air missile turrets.

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