Infections by trypanosomatid parasites cause Chagas disease, Human African Trypanosomiasis, and Leishmaniasis, affecting over 12 million people worldwide. Glycosomes, the unique peroxisome-related organelles of trypanosomes are essential for their survival, and hence their metabolic functions and biogenesis mediated by peroxins (PEX) are suitable as drug targets. Here we report on a comprehensive protein inventory of glycosomal membranes through advanced subcellular membrane protein profiling employing mass spectrometry. Our quantitative analysis resulted in the identification of 28 novel high confidence glycosomal membrane proteins. We validated four so far unknown glycosomal membrane proteins, including two tail-anchored (TA) proteins, a homolog of human peroxisomal PXMP4, and a Macrodomain containing protein. Using a structure-based approach, we identified one of the TA proteins as the long-sought Trypanosoma PEX15. Despite its low sequence similarity, Trypanosoma PEX15 exhibits structural and topological similarities with its yeast (Pex15) and human counterparts (PEX26). We show that PEX15 is an essential integral glycosomal membrane protein that interacts with PEX6. Accordingly, RNAi knockdown of PEX15 in bloodstream form trypanosomes demonstrates that it is essential for glycosome biogenesis and parasite survival. Considering the low degree of conservation with its human counterpart, PEX15 is a promising molecular target for drug development.