In arthropods, the cleavage of specific proteins by peptidases has pivotal roles in multiple physiological processes including oogenesis, immunity, nutrition, and parasitic infection. These enzymes are also key players in the larval development, and well-described triggers of molting and metamorphosis. In this work the peptidase complement throughout the larvae development of Penaeus vannamei was quantified at the transcript and activity level using qPCR and fluorogenic substrates designed to be hydrolyzed by class-specific peptidases respectively, providing a detailed identification of the proteolytic repertoire in P. vannamei larvae. Significant changes in the peptidase activity profile were observed. During the lecithotrophic naupliar instars, the dominant peptidase activity and expression derive from cysteine peptidases, suggesting that enzymes of this class hydrolyze the protein components of yolk as the primary amino acid source. At the first feeding instar, zoea, dominant serine peptidase activity was found where trypsin activity is particularly high, supporting previous observations that during zoea the breakdown of food protein is primarily enzymatic. At decapodid stages the peptidase expression and activity is more diverse indicating that a multienzyme network achieves food digestion. Our results suggest that proteolytic enzymes fulfill specific functions during P. vannamei larval development.