Protein glycosylation in human semen is crucial for spermatogenesis, maturation, sperm motility, capacitation, and fertilization. However, the site-specific information on N- and O-glycosylation in human semen has not been fully and accurately identified, particularly O-glycosylation. To address this, an integrated platform (termed GlycoIP) was developed for the simultaneous profiling of intact N- and O-glycopeptides from human semen. Although characterizing intact glycopeptides presents a challenge, their analysis can provide rich information about both the glycans and glycosites simultaneously. In this study, a total of 1,833 unique intact N-glycopeptides and 720 unique intact O-glycopeptides were reported based on GlycoIP. Much deeper and more accurate site-specific N/O-glycosylation information was revealed in this study, including 438 O-glycosites from 148 distinctive O-glycoproteins that may play important roles in human semen composition and function. In summary, GlycoIP provided a potentially useful method for the systematically profiling protein N/O-glycosylation in a single experiment. This lays the foundation for the functional studies of glycoproteins in male infertility.