We developed a novel method for analyzing d-limonene levels in adipose tissue. Fat samples were subjected to saponification followed by solvent extraction. d-Limonene in the sample extract was analyzed using gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring. Linear calibration curves were established over the mass range of 79.0-2529 ng d-limonene per 0.1g of adipose tissue. Satisfactory within-day precision (R.S.D. 6.7-9.6%) and accuracy (%difference of -2.7 to 3.8%) and between-day precision (R.S.D. 6.0-10.7%) and accuracy (%difference of 1.8-2.6%) were achieved. The assay was successfully applied to human fat biopsy samples from a d-limonene feeding trial.