AbstractIn recent years, the use of skeletal tissue as an alternative matrix in forensic toxicology has received new interest. In cases where extreme decomposition has taken place, analysis of skeletal tissue is often the only option left. In this article, a fully validated method is presented and the distribution of clomipramine, citalopram, midazolam, and metabolites after chronically administration is examined within skeletal tissue. Rats were chronically dosed with respectively clomipramine, citalopram, or midazolam. Extracts were quantitatively analyzed using liquid chromatography−electrospray ionization−tandem mass spectrometry (LC−ESI−MS/MS). Clomipramine, citalopram, and metabolites, respectively desmethylclomipramine and desmethylcitalopram are shown to be detectable in all bone types sampled. Midazolam and its metabolite α‐OH‐midazolam could not be detected. The absence of midazolam in extracts gives an indication that drugs with pKa values under physiological pH are badly or not incorporated in bone tissue. Bone and post‐mortem blood concentrations were compared. A range of different bone types was compared and showed that the concentration is strongly dependent on the bone type. In concordance with previous publications, the humerus shows the highest drug levels. Skeletal tissue concentrations found ranged from 1.1 to 587.8 ng/g. Comparison of the same bone type between the different rats showed high variances. However, the drugs−metabolite ratio proved to have lower variances (<20%). Moreover, the drugs−metabolite ratio in the sampled bones is in close concordance to the ratios seen in blood within a rat. From this, we can assume that the drugs−metabolite ratio in skeletal tissue may prove to be more useful than absolute found concentration.