The study objective is to develop approaches to the retrospective assay of tropicamide in biological fluids and hair. The study was performed using the substance of tropicamide. Sample preparation included hydrolysis with the following enzymes: papain, chymotrypsin, trypsin, chymopsin, and hyaluronidase. Extracts were analyzed using a gas chromatograph with mass selective detection Technologies (USA) 7890 A/5977 MSD. When modeling long-term use of tropicamide, male rats of white and brown natural color, about 6 months old and weighing 200—250 g, were used. Animals were injected with a tropicamide solution in the tail vein for 28 days at a dose of 40 mg/kg of body weight. After 28 days of administration of the tropicamide solution, daily urine and blood were collected, and hair was cut from the back and sides of the animal’s body. After another 28 days, hair samples were taken again. Within the first 6 hours after the last tropicamide dose, its blood concentration reached the maximum (191.6 μg/ml) and within 4 days decreased by 10 times; in the urine, within the first 24 hours, tropicamide level decreased from 627.7 to 489.9 μg/ml, then for 2—3 days it remained approximately at the same level. From day 4, the tropicamide concentration significantly decreased, and on days 11—12, it was not detected in the urine. After 4 weeks, the tropicamide content in the hair was at the level of quantification (1.25—2.20 ng/mg) and could be detected only by sample preparation by enzymatic hydrolysis with papain. Thus, the developed and validated methods for the enzymatic hydrolysis of biological fluids and hair allowed retrospective studies of biological fluids and hair with high reliability.