Proteome analysis using human serum is a technological advancement that will enable the discovery of novel biomarkers and biomarker patterns of various human diseases. Although proteome analysis using serum has potential in disease prevention, early diagnosis and treatment of diseases, and evaluation of pharmacotherapies, this technology is still in its infancy. Thus, we sought to develop an advanced method of conducting proteome analysis on human serum. In this study, we report the development of the semi-comprehensive protein analytical technique, which involves the systematic use of iTRAQ labeling, HPLC, nano-LC and MS. We compared the composition of the serum proteome in males and females using this technique and detected gender-based differences in serum protein composition. This technology will enable the generation of databases that may ultimately lead to the discovery of specific biomarkers or biomarker patterns of various diseases.