An LC method was developed and validated in order to quantify the saponin purified fraction named QB-90 obtained from the aqueous extracts of Quillaja brasiliensis leaves. Previously, QB-90 was shown to be active as adjuvant in an experimental vaccine for herpesvirus bovine type 1 in mice. The analysis was performed using an RP-8 column with acetonitrile:water isocratic elution at 214 nm. The standard curve for QB-90 was linear over the range of 0.8-10 µg mL(-1) (r = 0.9996). The aqueous extract showed linear response in the range of 50-1000 µg mL(-1) (r = 0.9996). The proposed method showed adequate repeatability and intermediary precision. The limits of detection (LOD) and quantification (LOQ) were 0.074 and 0.248 µg mL(-1), respectively. This method was applied successfully to analyse QB-90 organ-specific distribution in field stands of Q. brasiliensis and in laboratory-grown seedlings. Leaves from young plants accumulated higher QB-90 amounts than leaves from adult trees.