The application of mass spectrometry based proteomics to sperm biology has greatly accelerated progress in understanding the molecular composition and function of spermatozoa.Here we apply high-throughput mass spectrometry proteomic analysis to characterize sperm composition in Manduca sexta, the tobacco homworm moth, which produce heteromorphic sperm, including one fertilization competent (eupyrene) and one incompetent (apyrene) sperm type.