SK-MEL-2 cell line belongs to series of melanoma cell lines established from patient-derived tumor samples, which is usually applied in the melanin-related mechanism studies. TMT-labeling quantitative proteomics which is the state-of-art LC-MS/MS technique for protein quantitation, in this study, was applied to reveal the overall response of proteins involved in the polymyxin B-induced melanogenesis in SK-MEL-2 cells, in order to provide a possible clue at the cellular level for understanding mechanisms of polymyxin B-induced skin hyperpigmentation in clinics.