The protozoan parasite Trypanosoma brucei causes the disease Human African Trypanosomiasis (HAT) and the cattle wasting disease, Nagana. To avoid difficulties associated with protein alignments, we used small molecules that perturb trypanosome biology and then identified proteins within the affected biological pathways. The kinase inhibitor AEE788 selectively blocks transferrin endocytosis without affecting uptake of bovine serum albumin. We have used hypothesis-generating phospho-proteomics to identify the protein Tb427tmp.160.4770 as an AEE788-pathway protein. Using RNAi, we establish Tb427tmp.160.4770 as a selective regulator of transferrin endocytosis. We used comparative phospho-proteomics after knockdown of Tb427tmp.160.4770 to identify Tb427tmp.160.4770 pathway proteins, five of which were also identified as AEE788 pathway proteins. We hypothesize that the AEE788 and Tb427tmp.160.4770 pathway proteins are selective modulators of transferrin uptake in the African trypanosome. These studies highlight the power of small molecules as tools for identifying new and regulatorygenes involved in transferrin endocytosis pathway in T. brucei.