Diffuse reflectance spectroscopy (DRS) can be used to estimate oxygen saturation (SO2) of hemoglobin and blood fraction (fB) in brain tissue. The aim of the study was to investigate the SO2 and fB in different positions along deep brain stimulation (DBS) trajectories and in specific target regions using DRS and a novel algorithm. DRS measurements were done at 166 well-defined anatomical positions in relation to stereotactic DBS-implantation along 20 trajectories toward 4 DBS targets (STN, Vim, GPi and Zi). The measurements were dived into groups (gray, white and light gray matter) related to anatomical position, and DBS targets, before comparison and statistical analysis. The median SO2 in gray, white and light gray matter were 52%, 24% and 20%, respectively. Median fB in gray matter (3.9%) was different from values in white (1.0%, p < 0.05) and light gray (0.9%, p < 0.001) matter. No significant difference in median SO2 and fB was found between DBS target regions. The novel algorithm allows for quick and reliable estimation of SO2 and fB in human brain tissue.