There is the present-day tendency toward prescribing atypical neuroleptics for the management of neurologic and psychic disorders. Alimemazine appears to be very frequently used for this purpose due to the broad spectrum of its actions. At the same time, cases of alimemazine poisoning with the fatal outcome have been described. The objective of the present study was determine alimemazine in the biological fluids from the laboratory animals under the acute poisoning conditions. The experiments were carried with the use of the Wistar rats having 200 g body weight. Alimemazine was isolated from their biological fluids (blood plasma and urine) using the liquid-liquid extraction techniques developed specially for the purpose of this study. Alimemazine was extracted and quantitatively determined by HPLC and HPLC/MS. The method for the isolation of alimemazine from the urine and blood plasma is described. The results of the study give evidence that the maximum amount of the substance of interest can be extracted from the blood plasma within 1 hour after the administration of the toxic dose of alimemazine and within 2 hours after the administration of its therapeutic dose. The maximum amount of alimemazine in the urine is found within 3 hours after the administration of its therapeutic dose to the laboratory animals. It is concluded that the proposed methods for the extraction of alimemazine from the biological fluids can be included in the scheme of the chemical toxicological analysis of this substance.