Using time-resolved spectroscopy, we have developed an experimental approach to obtain the absolute changes in concentration in the scattering medium of living tissues. The time-resolved Beer-Lambert equation can be applied to living tissue due to the fact that the optical attenuation by absorption can be separated from that by scattering, and the intensity of the light along the non-linear scattered optical path is exponentially attenuated by the absorption. Based on the above, the absolute concentration of haemoglobin as well as oxygen saturation in the rat head can be determined in situ under various respiratory conditions where multiwavelength measurements were performed. The optically assessed values agree with those determined directly by the gas analysis of our previous report. The present method is very simple and therefore opens up wide applications for time-resolved spectrophotometry in clinical medicine as a technique for quantitative near-infrared oxygen monitoring.