Background and Aims: Carnivorous plants have developed strategies to enable growth in nutrient-poor soils. For the genus Nepenthes, this strategy represents producing pitcher-modified leaves that can trap and digest various prey. These pitchers produce a digestive fluid composed of proteins, including hydrolytic enzymes. The focus of this study was on the identification of these proteins. Methods: In order to better characterize and have an overview of these proteins, digestive fluid was sampled from pitchers at different stages of maturity from five species of Nepenthes (N. mirabilis, N. alata, N. sanguinea, N. bicalcarata and N. albomarginata) that vary in their ecological niches and grew under different conditions. Three complementary approaches based on transcriptomic resources, mass spectrometry and in silico analysis were used. Key Results: This study permitted the identification of 29 proteins excreted in the pitchers. Twenty of these proteins were never reported in Nepenthes previously and included serine carboxypeptidases, α- and β-galactosidases, lipid transfer proteins and esterases/lipases. These 20 proteins display sequence signals allowing their secretion into the pitcher fluid. Conclusions:Nepenthes pitcher plants have evolved an arsenal of enzymes to digest prey caught in their traps. The panel of new proteins identified in this study provides new insights into the digestive process of these carnivorous plants.