Untargeted metabolomic studies are used for large-scale analysis of endogenous compounds. Due to exceptional long detection windows of incorporated substances in hair, analysis of hair samples for retrospective monitoring of metabolome changes has recently been introduced. However, information on the general behavior of metabolites in hair samples is scarce, hampering correct data interpretation so far. The presented study aimed to investigate endogenous metabolites depending on hair color and along the hair strand and to propose recommendations for best practice in hair metabolomic studies. A metabolite selection was analyzed using untargeted data acquisition in genuine hair samples from different hair colors and after segmentation in 3 cm segments. Significant differences in metabolites among hair colors and segments were found. In conclusion, consideration of hair color and hair segments is necessary for hair metabolomic studies and, subsequently, recommendations for best practice in hair metabolomic studies were proposed.