Plasma lipoproteins, such as high‐density lipoprotein (HDL), can serve as carriers for a wide range of proteins that are involved in processes such as lipid metabolism, thrombosis, inflammation and atherosclerosis. The identification of HDL‐associated proteins is essential with regards to understanding these processes at the molecular level. In this study, a combination of proteomic approaches including 1‐DE and 2‐DE MALDI‐TOF, isotope‐coded affinity tag and Western blot analysis were employed to identify proteins associated with human HDL. To minimize potential losses of HDL‐associated proteins during isolation, a one‐step ultracentrifugation technique was applied and the quality of purified HDL was confirmed by nephelometry, high‐performance gel chromatography, and Western blot analysis. MS analysis revealed the presence of 56 HDL‐associated proteins including all known apolipoproteins and lipid transport proteins. Furthermore, proteins involved in hemostasis and thrombosis, the immune and complement system were found. In addition, growth factors, receptors, hormone‐associated proteins and many other proteins were found to be associated with HDL. Our approach thus resulted in the identification of a large number of proteins associated with HDL. The combination of proteomic technologies proved to be a powerful and comprehensive tool for the identification of proteins on HDL.