Safe and accurate placement of pedicle screws remains a critical step in open and minimally invasive spine surgery. The diffuse reflectance spectroscopy (DRS) technique may offer the possibility of intra-operative guidance for pedicle screw placement. Currently, Magnetic Resonance Imaging (MRI) is one of the most accurate techniques used to measure fat concentration in tissues. Therefore, the purpose of this study is to compare the accuracy of fat content measured invasively in vertebrae using DRS and validate it against the Proton density fat fraction (PDFF) derived via MRI. Chemical shift-encoding-based water-fat imaging of the spine was first performed on six cadavers. PDFF images were computed and manually segmented. 23 insertions using a custom-made screw probe with integrated optical fibers were then performed under cone beam computer tomography (CBCT). DR spectra were recorded at several positions along the trajectory as the optical screw probe was inserted turn by turn into the vertebral body. Fat fractions determined via DRS and MRI techniques were compared by spatially correlating the optical screw probe position within the vertebrae on CBCT images with respect to the PDFF images. The fat fraction determined by DRS was found to have a high correlation with those determined by MRI, with a Pearson coefficient of 0.950 (P< 0.001) as compared with PDFF measurements calculated from the MRI technique. Additionally, the two techniques were found to be comparable for fat fraction quantification within vertebral bodies (R2 = 0.905).