In forensic toxicology, when extended time has elapsed before discovery of the body, the most commonly analyzed specimens are often degraded or not available at all due to decomposition. In this case, skeletal tissue may be the only specimen left. Nevertheless, very limited research is found on the drug disposition in bone, making toxicological interpretation very difficult. Since methadone is linked to almost 50% of drug abuse fatalities in Belgium, an easy extraction and quantification method was developed and validated to investigate the distribution pattern of methadone and its metabolites in skeletal tissue after chronic dosing. In this study, Wistar rats were administered a subcutaneous daily methadone dose of 3 mg/kg for 139 days. After dissection, single whole bones or bone parts underwent a methanolic extraction. The final extract was analyzed using LC-ESI(+)-MS-MS for methadone, EDDP and EMDP. Methadone and its metabolites were proven to be detectable and quantifiable in skeletal tissue of chronically dosed rats using a fast and easy methanol extraction. Within bone, comparison showed that bone marrow yields the highest concentration. Trabecular bone also showed to be the best type of bone tissue for sampling. Between bone comparison, proved the humerus to be the best bone type for sampling. The concentrations found in tibiae and humeri appeared to be dose dependent for methadone with a variance of <9%. However, for other bones the variance in methadone concentration ranged from 24 to 32%. A possible explanation is seen in the lower vascularization of these bones. For the metabolites, no correlation was seen. This could be explained by the highly inter-individual metabolism of methadone. However, skeletal tissue concentration showed no correlation to blood for methadone nor its metabolites. Using the developed method, quantitative information about methadone after chronic administration was only found in the humeri and tibiae.