Relevance of the study: The quantification of endogenous substances is an important task in experimental and clinical pharmacology. In the case of working with endogenous compounds, certain difficulties arise. The main one is the impossibility of obtaining the same biomatrix without endogenous compounds for use as standard solutions in the construction of calibration curves. Purpose: The aim of our study was to develop a mathematical methodology for calculating the concentration of endogenous compounds in biological objects measured by chromatography, which allows us to obtain a statistically reliable interval estimation of the concentration of endogenous compounds. Materials and methods: To implement the computational part of the proposed algorithm, the Mathcad engineering calculation program version 15.0 from PTC (Parametric Technology Corporation) is used, which operates under the family of Windows operating systems (XP, 7, Vista, 8). Main results: A mathematical methodology has been developed for calculating the concentration of endogenous compounds in biological objects measured by chromatography, which allows one to obtain a statistically reliable interval estimate of the concentration of endogenous compounds. A feature of this technique is the use of an exclusively analyzed bioobject for quantitative determination of endogenous substances, without the use of so-called “pure” bioobjects for calibration curves, as well as expensive deuterated analogs of markers – substrates of CYP450 isoenzymes and their metabolites. This allows you to maintain the original biomatrix effect when removing the chromatogram. A statistical apparatus is also used to eliminate possible gross errors. To confirm the reliability of the developed method of quantitative determination, the convergence of the results obtained by this method using deuterated standards is established. Conclusions: A mathematical methodology has been developed for calculating the concentration of endogenous compounds in biological objects measured by chromatography, which allows one to obtain a statistically reliable interval estimate of the concentration of endogenous compounds. A feature of this technique is the use of only the analyzed bioobject for quantitative determination of endogenous substances, which allows you to maintain the original biomatrix effect when removing the chromatogram.