Abstract: The aim of current investigation was the validation of HPLC method with UV- detection for identification and determination of Yohimbine by usingcolumn Spherisorb ODS RP C18 (250 mm x 4.6 mm x 5 μm), temperature: 25 oC, mobile phase: methanol : acetonitrile : water = 70 : 20 : 10 v/v, flow rate: 2 ml/min., UV-detection: λ = 270 nm. HPLC method was validated in accordance of linearity, litmit of detection, limit of quantitation, accuracy, precision. Linear dependence between concentration and peak area in range: 5.10–7 g/ml ÷ 1.10–5 g/ml was proved by regression equation: y = 94517560831 . x + 25119. LOD = 5.44 10-7 g/ml; LOQ = 1.65.10-6 g/ml. Accuracy was presented by the degree recovery R [%] ± RSD [%]: 98.68 % ± 2.03 %. For estimation of precision all data for Yohimbine content are included in confidence interval: 9.6 mg ÷ 10.14 mg.