Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, and flagella and needle-shaped nuclei with highly compacted genomes are formed. As transcription takes place mainly in spermatocytes and transcripts relevant for post-meiotic sperm development are translationally repressed for days, we comparatively analysed the proteome of larval testes (only germ cell stages before meiotic divisions), testes of 1-2-day-old pupae (germ cell stages before meiotic divisions, meiotic and early spermatid stages) and adult flies (germ cell stages before meiotic divisions, meiotic and early spermatid stages, late spermatids and sperm). We identified 6,171 proteins; 61 proteins were detected solely in one stage and are thus enriched, namely 34 in larval testes, 77 in pupal testes and 214 in adult testes. To substantiate our mass spectrometric data, we analysed the stage-specific synthesis and importance for male fertility of a number of uncharacterized proteins. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein and was present in the transition phase from a histone-based to a protamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg were sterile due to the lack of sperm in the seminal vesicles. Our catalogue of proteins of the different stages of testis development in D. melanogaster will pave the road for future analyses of spermatogenesis.