We report a second derivative multispectral algorithm for quantitative assessment of cutaneous tissue oxygen saturation (). The algorithm is based on a forward model of light transport in multilayered skin tissue and an inverse algorithm for reconstruction. Based on the forward simulation results, a parameter of a second derivative ratio (SDR) is derived as a function of cutaneous tissue . The SDR function is optimized at a wavelength set of 544, 552, 568, 576, 592, and 600 nm so that cutaneous tissue can be derived with minimal artifacts by blood concentration, tissue scattering, and melanin concentration. The proposed multispectral imaging algorithm is verified in both benchtop and in vivo experiments. The experimental results show that the proposed multispectral imaging algorithm is able to map cutaneous tissue in high temporal resolution with reduced measurement artifacts induced by different skin conditions in comparison with other three commercial tissue oxygen measurement systems. These results indicate that the multispectral imaging technique has the potential for noninvasive and quantitative assessment of skin tissue oxygenation with a high temporal resolution.