The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localisation of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with an alkyne-tagged myristic acid analogue (“YnMyr”), enabling the capture of lipidated proteins in insect (PCF) and host (BSF) life stages of T. brucei. We further compare this with a longer chain palmitate analogue (“YnPal”) to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors (Cpds 1 and 2) and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites.