<p>Polypore fungi represent a promising source of biologically active triterpenoid and steroid compounds; however, their quantitative determination in the raw material is complicated by the lack of reference standards and the variety and structural similarity of the compounds. The present study developed and validated a spectrophotometric method based on the Liebermann–Burchard reaction for the determination of total triterpenes and steroids in polypore extracts. The optimal analytical conditions were established as follows: CHCl₃ as the extractant, 1:1 extract-to-reagent ratio, analytical wavelength of 665 nm, and reaction time of 90 min. The method demonstrated satisfactory specificity, accuracy (recoveries of 100.6–108.6%), precision (RSD ≤ 3.4%), and linearity in the range 31.25–1000 μg/mL (R² &gt; 0.99). The results confirmed the applicability of the developed approach for standardization of fungal raw materials and quality control of polypore-based products.</p>