Updated project metadata. Human spermatozoa proteomics exposed to some physical, biological or chemical stressors is being explored. However, there is a lack of systematic comparative study on protein extraction methods to achieve in-depth coverage. Meanwhile, it is not clear if antibiotics can regulate proteins to affect sperm quality. Here we systematically compared and optimized the sperm protein extraction methods. It showed that the UA_Ultrasonication extraction method can make more kinds of protein to obtain higher protein extraction rate. Applying the optimized method, a largest human sperm proteome (5685 protein groups) from healthy human sperm samples was described. Besides, we continually investigated for the first time the differential sperm characteristics between medication (amoxicillin and clarithromycin) period and withdrawal period’s sperm samples obtained from a patient with Helicobacter pylori infection using semen examination, morphological analysis, bioinformatic analysis and data-independent acquisition tandem mass spectrometry (DIA-MS/MS). The longitudinal study result indicated that antibiotics can disrupt proteome expression in sperm, leading quantitative coverage of 159 differentially expressed proteins. The reliability of the results was verified by four proteins (ACTB, SCRN2, FDFT1, PLPP1) quantified by western blot. These proteins were mainly located at cytosol, involved in phosphorylation process, adenosine triphosphate (ATP) binding and metabolic pathways using bioinformatic analysis. This work provides an optimized extraction method to characterize the in-depth human sperm proteome and to extend its clinical applications. In addition, antibiotics should be avoided when it is necessary to preserve sperm quality for reproductive purpose.