Simple, rapid and sensitive spectrophotometric method is developed for the determination of glucose in pure form and in human embryos culture medium. This method is based on the reduction of Triphenyltetrazolium chloride (TTC) by glucose in 4.0 mM NaOH medium to produce a colored stable precipitate. The product was dissolved in 35−45 % ethanol and absorbance was measured at 480 nm. All experimental conditions affecting the development and stability of color were carefully studied and optimized. The calibration graphs are rectilinear in the concentration range of 14.59−29.19 µg mL-1for glucose in pure form and of 18.16−39.95 µg mL-1 for glucose in a synthetic culture medium mixture. Sandell sensitivity (S), molar absorptivity (ε), correlation coefficient (R2), regression equation, limit of detection (LOD), and limit of quantification (LOQ) are calculated in both cases. The low values of standard deviation (0.0173−0.3958) for glucose in pure form and (0.1440−0.5430) for glucose in the synthetic culture medium mixture, and the relative standard deviation (0.0619−2.009 %) for glucose in pure form and (0.4920−2.750 %) for glucose in the synthetic culture medium mixture reflect the accuracy and precision of the proposed method. The method was applied for the determination of glucose in human embryos culture medium; which contains glucose as one of its main components. The recovery values, standard deviation and relative standard deviation of glucose determination in the actual human embryos culture medium are found to be 100.0 %, 0.1891 and 0.3885 %, respectively. The formed reduction product was extracted in chloroform. The proposed reaction pathway of glucose with TTC reagent was postulated and confirmed by separation of the reaction product which was proved to be 1,3,5-triphenylformazan (TPF). The structure of the separated reaction product was confirmed by elemental analysis, FT-IR, and 1H-NMR.