Human growth hormone (hGH) is a naturally occurring, 191 amino acid peptide hormone secreted by the pituitary gland with anabolic and growth-promoting activity. Since the increased availability of rhGH in the late 1980s for the treatment of hGH-deficient patients, rhGH has become a drug of abuse in sport and it is prohibited by the World Anti-Doping Agency (WADA). Currently, there is no method available that can clearly discriminate between GH user and nonuser due to difficulties in direct differentiation between native and recombinant iso-forms of hGH, short half-life in circulation, and not enough biomarkers reported. We have identified new GH-depended plasma biomarkers by two-dimensional difference gel electrophoresis (2-D DIGE) and Isobaric tags for relative and absolute quantitation (iTRAQ) analyses which have longer half-life than rhGH itself. We have validated the identities of candidate markers by using orthogonal methods such as western blotting and selected reaction mass spectrometric (SRM) analysis. By combining bio-markers reported by others and new markers that we have identified in our work may provide us an opportunity to develop a better rhGH abuse detection method in sports or monitoring changes in plasma proteome after GH-administration in clinical trials.