Steroid hormone analysis is widely used in health- and stress-related research to get insights into various diseases and the adaption to stress. Hair analysis has been used as a tool for the long-term monitoring of these steroid hormones. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous identification and quantification of seven steroid hormones (cortisone, cortisol, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, progesterone) in hair. Cortisol, cortisone, androstenedione, testosterone and progesterone were detected and quantified in authentic hair samples of different individuals. Significantly higher concentrations for body hair were found for cortisone and testosterone compared to scalp hair. Furthermore, weak correlations for the majority of steroids between scalp and body hair indicate that body hair is not really suitable as alternative when scalp hair is not available. The influence of hair pigmentation was analyzed by comparing pigmented to non-pigmented hair of grey-haired individuals. The results showed no differences for cortisol, cortisone, androstenedione, testosterone and progesterone concentrations (p > 0.05) implying that hair pigmentation has not a strong effect on steroid hormone concentrations. Correlations between hair and nail steroid levels were also studied. Higher concentrations of cortisol and cortisone in hair were found compared to nails (p < 0.0001). No significant correlation for cortisone, cortisol, androstenedione, testosterone and progesterone concentrations were found between hair and nails. These results demonstrate that matrix-dependent value ranges for hair and nail steroid levels should be established and applied for interpretation.