Simple and effective procedure was developed for solid-phase extraction of 1,25(OH)2D (calcitriol) from blood serum or blood plasma using mini-columns "Silica-Cart C18" which enabled to purify the hormone preparation to the state suitable for its subsequent evaluation by means of RRA and RIA. Separation of 1,25(OH)2D from 25-OHD and purification from lipophilic impurities were carried out on the single mini-column where solvents with gradually decreased polarity were used: water, methanol-water (7:3), hexane-chloroform (9:1), hexane-isopropanol (99:1), hexane-isopropanol (85:15). Concentration of 1,25(OH)2D was adequately estimated in blood serum of patients with chronic kidney insufficiency as well as in blood serum of vitamin D deficient animals.