Incidence of suicidal attempts presents an explanation for the high prevalence of quetiapine (QTI) in forensic cases. Thus, the interpretation of its concentrations in biological specimens is needed, but in forensic toxicology, potential postmortem changes such as instability of the target drugs should be taken in consideration. High-performance liquid chromatography (HPLC) method has been developed for determination of QTI. This method was based on reversed phase (RP)-HPLC separation of QTI on a C-18 column (150 mm × 4.6 mm, 5 μm) with elution system of acetonitrile—methanol—0.025 M phosphate buffer (pH 2.5), containing 1 mL TEA in each 250 mL, in a ratio of 40:30:30%, v/v, at the flow rate of 1.2 mL min−1 using mirtazapine as internal standard (IS). The proposed method was applied to the determination of QTI in plasma in presence of coadministered drugs. The application of the proposed method was extended for long-term stability study of two different concentration levels of QTI in the whole blood.