Forensic chemical examination is carried out in order to isolate, identify and quantify toxic, narcotic, psychotropic and potent substances in the organs and fluids of the human body. The present study we researched the process of extraction of 2,3-benzodiazepine - tofisopam. There have been cases of tofisopam poisoning. And there are no descriptions of tofisopam isolation from biological fluids in the literature. Liquid extraction, an effective and commonly used method of isolation in the chemical-toxicological analysis, is used when extracting substances from blood, lymph, saliva, urine and stomach washings. The process of isolation may be influenced by the nature of the organic solvent, pH, the presence of an electrolyte, time and frequency of extraction. We have studied the effect of each factor on tofisopam isolation from aqueous solutions. It has been found that the optimal organic solvent for the extraction of the solutions is dichloroethane, which extracts maximum amount of the analyte at pH = 5.0. Saturated sodium sulphate has a salting-in effect for tofisopam. The optimum conditions for tofisopam isolating are 7 minutes with double extraction. Based on the research method of tofisopam isolating the model mixture of urine was developed. We found that this model urine mixture isolates 66.51-67.9l % of tofisopam. We performed validation assessment of the developed method for evaluation of precision and accuracy. The assessment showed the suitability of techniques for the analysis.