Abstract: Simultaneous reactions of aniline on the aldehyde groups and ammonium molybdate on the ortho-diphenolic groups of gossypol were studied in tartaric acid buffer solution. The results indicated in the range 300-520 nm that the spectrum of dianilino-dimolybdate of gossypol complex obtained presents two peaks of absorption at 340 and 450 nm whereas the dianilino-gossypol and the dimolybdate of gossypol present one peak at 444 and 420 nm, respectively. The pH effect on the intensity of the absorption indicated that the optimum pH was 6.5. Gossypol is the only compound in the cottonseed extract having two peaks of absorption in the range 300-520 nm. The calibration curve was linear over the range 0.634-4.953 ppm (r2 = 0. 9949) with the detection limit of 0.474 ppm. The stability of the complex was 90 min. The method was sensitive, specific and accurate for the determination of total gossypol in glanded cottonseed, crude, neutral and refined glanded cottonseed oil.