Melanin in the skin can be divided into eumelanin and pheomelanin subtypes. Simultaneous quantification of these subtypes could clarify their relation to skin type and skin cancer development. We describe a novel, sensitive liquid chromatography-tandem mass spectrometry method to quantify two eumelanin markers, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), and two pheomelanin markers, thiazole-4,5-dicarboxylic acid (TDCA) and thiazole-2,4,5 tricarboxylic acid (TTCA), performed in a single run using the same biopsy. Volunteers with either Fitzpatrick skin type (FST) I/II or III/IV (n = 30) each provided a 4-mm punch biopsy from the buttock. Upon analysis, the FST I + II group had significantly less of all four melanin biomarkers (PTCA, 0.75 ng/mm2 ; PDCA, 0.08 ng/mm2 ; TTCA, 0.24 ng/mm2 ; and TDCA, 0.10 ng/mm2 ) versus the FST III + IV group (PTCA, 4.89 ng/mm2 ; PDCA, 0.22 ng/mm2 ; TTCA, 2.61 ng/mm2 ; and TDCA, 0.72 ng/mm2 ), p ≤ 0.003. We find that this new LC-MS/MS method is sensitive enough to quantify eumelanin and pheomelanin markers even in the lightest skin types.