We present a method for simultaneous measurement of optical absorption coefficients for skin (μas) and muscle (μam) tissues using spatially resolved near-infrared spectroscopy (SRS). A novel calculation algorithm was developed to determine the absorption coefficients of superficial and deep layers within a three-layered structure using Monte Carlo simulation. A method for measuring the skin and muscle absorption coefficients was proposed based on this algorithm. In vitro experiments with tissue-like phantom and in vivo tests were performed using the SRS system with four separate detectors. The results show that the absorption coefficients for both skin and muscle tissues were obtained accurately.