Melanins are difficult to characterize because of their intractable chemical properties, the heterogeneity in their structural features, and the lack of methods to split melanin polymers into monomer units. To overcome this difficulty, we developed a rapid and sensitive method for quantitatively analyzing eumelanin and pheomelanin in biological samples that is based on the formation of pyrrole-2,3,5-tricarboxylic acid and/or aminohydroxyphenylalanine followed by HPLC determination. The method has been applied to the study of melanogenesis. The results summarized in this review are: 1) Biochemical studies show that in the process of mixed melanogenesis, cysteinyldopas are produced first, which are then oxidized to give pheomelanin; following cysteine depletion, eumelanin is then deposited on the preformed pheomelanin. 2) In vitro and in vivo studies show that tyrosinase activity is the most important factor that regulates the switch of melanogenesis, with lower tyrosinase activities favoring pheomelanogenesis; further suppression of melanogenesis results in a lack of pigment production. 3) In cultured melanocytes, the concentrations of tyrosine and cysteine, and their ratio in the medium, are important in determining the concentrations of eumelanin and pheomelanin produced and their ratio in the cells. In conclusion, our HPLC microanalytical method for characterizing eumelanin and pheomelanin has become a useful tool for the study of melanogenesis.