Abstract Adipose tissue is a complex biological matrix that necessitates several pre-analytical preparation steps to separate drugs and metabolites from the lipophilic matrix. A novel, sensitive, and specific gas chromatographic-mass spectrometric (GC-MS) method for the determination of cocaine (metabolites), methadone, and morphine in postmortem adipose tissue was developed, optimized, and validated. The method involves the aqueous acid extraction of analytes, alkalinization of the extract, solid-phase extraction with chloroform, and derivatization with BSTFA before GC-MS analysis. Deuterated compounds were used as internal standards for determination and quantification of analytes. Limits of detection were 0.005 µg/g for cocaine and cocaethylene, 0.02 µg/g for benzoylecgonine, 0.01 µg/g for ecgoninemethylester, 0.005 µg/g for methadone, and 0.01 µg/g for morphine. Linearity ranged from 0.1 to 1.000 µg/g for all analytes. Intra- and interday accuracy ranged from 70.6 to 105%, and intra- and interday precisions were less than 8.2% and 8.6%, respectively, for all analytes. The method showed a good recovery