The Chromosome-Centric Human Proteome Project aims to identify proteins classed as « missing » in the neXtProt knowledgebase. In this article, we present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using a range of protein extraction procedures and LC-MS/MS analysis, we detected a total of 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through a combination of LC-MS/MS and LC-PRM analysis, data mining and immunohistochemistry, we were able to confirm the expression of 206 missing proteins (PE2-4) in line with current HPP guidelines (version 2.0). Parallel Reaction Monitoring (PRM) acquisition combined with synthetic heavy labeled peptides was used to target 36 « one-hit wonder » candidates selected on the basis of prior PSM assessment. Of this subset of candidates, 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for a further 16 missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could potentially be valuable markers with applications in fertility assessment. Strong evidence was also found for the existence of 4 proteins labeled as "uncertain" (PE5); the status of these proteins should therefore be re-examined. Our results show how the use of a range of sample preparation techniques combined with MS-based analysis, expert knowledge and complementary antibody-based techniques can produce data of interest to the community. All MS/MS data are available via ProteomeXchange under identifier PXD003947. In addition to contributing to the Chromosome-Centric Human Proteome Project, we hope the availability of these data will stimulate the continued exploration of the sperm proteome.