Abstract: Spectrometric methodology for non-invasively obtaining optical spectra from heterogeneous material for the identification and quantification of constituent compounds. There is provided a transient or steady state subsurface thermal gradient spectroscopic methodology for obtaining in vivo optical spectra relating to the concentration of .eta. analytes at depths to around 330 microns in human tissue, and for determining that concentration from the spectra. The methodology is employable on a wide variety of spectrometric devices, and enables: a real time determination of both surface and reference intensities; a fast, efficient calibration of the spectrometric device; and results in the provision of an analytical parameter which avoids the measurement of the optical path length to enable the extremely accurate calculation of a ratio of concentrations of .eta. analytes in the system under analysis.