This project investigates the impact of N-myristoyltransferase (NMT) inhibition on the myristoylated proteome of Trypanosoma cruzi and human cardiomyocytes in the context of Chagas disease. Human AC16 cardiomyocytes were infected with T. cruzi (CL Brener strain) and treated with the reference NMT inhibitor DDD85646 or vehicle control. Myristoylated proteins from intracellular amastigotes, extracellular trypomastigotes, and host cells were metabolically labeled with an azide-functionalized myristic acid analogue, enriched by click-chemistry, digested, and labeled with TMTsixplex reagents for multiplexed quantification. Labeled peptide fractions were analyzed by nanoLC–MS/MS on a Vanquish Neo UHPLC coupled to an Orbitrap Exploris 240 operated in data-dependent acquisition mode. The resulting dataset was used to quantify stage-specific changes in parasite and host myristoylated proteins upon TcNMT inhibition and to identify pathways related to vesicular trafficking, lipid metabolism, and signal transduction that are selectively perturbed in the parasite.