The determination of phosphate in human embryos' culture medium is the main target of this manuscript using novel mixed reagents. This research also aimed to perform comparative evaluation of spectrophotometrically determined phosphate in synthetic and in actual human embryos' culture medium. It depents on the reaction of phosphate with mixed colorimetric reagents. The molybdenum blue formed attained a λmax of 825 nm and molar absorptivity of 2.190 x 103 L mol-1 cm-1. The product of the reaction in case of adding potassium hydrogen tartrate to the reagent showed a λmax at 830 nm and the molar absorptivity of molybdenum blue was 2.833 x 104 L mol-1 cm-1. So, antimony potassium tartrate was added to the reagent to increase the reaction rate in order to be able to make determination at the maximum absorbance of the product and subsequently increase the sensitivity of the method. This reduced the time of reaction to 5−10 min and wavelength at the maximum absorbance was shifted to be 896 nm with molar absorptivity 1.797 x 104 L mol-1 cm-1. Tracing the reaction between sodium dihydrogen phosphate and ammonium molybdate under the optimum conditions led to the construction of a calibration curve. The concentration range of the calibration graph was found to be 3.183−17.5 µg mL-1 and correlation coefficient of the data obtained was 0.999. The method was applied to a synthetic mixture containing some components of the human embryos' culture medium without interference. This leads to the successful application of the suggested procedures to the spectrophotometric micro-determination of phosphate content in actual human embryos' culture medium; which was in a good matching with the data of determining phosphate in the same media in literature.