To estimate the concentrations of melanin and blood and the oxygen saturation in human skin tissue, we propose a method using a multiple regression analysis aided by a Monte Carlo simulation for diffuse reflectance spectra from the skin tissue. By using the absorbance spectrum as a response variable and the extinction coefficients of melanin, oxygenated hemoglobin, and deoxygenated hemoglobin as predictor variables, the multiple regression analysis gives regression coefficients. The concentrations of melanin and blood are determined from the regression coefficients using conversion vectors that are estimated numerically in advance, while the oxygen saturation is obtained directly from the regression coefficients. Numerical and experimental investigations were performed for layered skin tissue models and phantoms. Measurements of human skin were also carried out to monitor variations in the melanin and blood contents and oxygenation during cuff occlusion. The results confirmed the usefulness of the proposed method.