The causative agents of animal trypanosomosis are various species of protozoan parasites belonging to the genus Trypanosoma, among which T. congolense and T. evansi are the major pathogenic species. The extra cellular position of the trypanosomes implies to consider both the parasite and its excretedsecreted factors in the course of the physiopathological processes. Proteome (i.e. parasite constitutive proteins) and secretome (i.e. naturally excreted-secreted proteins) analysis need to be conducted in parallel to identify key trypanosome¿s proteins potentially involved in both virulence and pathogenicity. We developed and standardised a method to produce purified proteome and secretome of the two sub-genus Trypanozoon and Nannomonas. The protocol is based on the production of trypanosome bloodstream forms by infection of naturally immunosupressed rodents (Nude/SOPF®) and incubated in a defined secretion medium that mimics the blood biochemical environment deprived of cells and macromolecules. Supernatants representing secretome are separated from parasites pellets representing proteome by centrifugation and filtration and both are conditioned for further analysis in two-dimensional gel electrophoresis and mass spectrometry. The defined secretion medium appears to reduced expression of stress proteins. Two-dimensional difference gel electrophoresis (2-D DIGE) analysis of secretomes in particular confirmed both the differences observed in 1-D gels and the high reproducibility between secretome batches of a same trypanosome strain. The molecular identification of differentially expressed trypanosomes molecules correlated with either the virulence process or the pathogenicity will provide new potential molecular targets.