Cefotaxime is considered as one of the semi-synthetic, 3rd generation, cephalosporin antibiotics that have been utilized for treating a lot of infections resulting from different organisms. The technique of hydrophilic chromatography interaction (HILIC) has increased the accuracy and sensitivity concerning other methods, particularly spectrophotometer. The objective of this article was to introduce a simple method for the estimation of cefotaxime in pure and pharmaceutical injection forms and study the separation mechanism of cefotaxime. HILIC mode achieved excellent separation under chromatographic conditions on a HALO ® HILIC 2.7 column (100 mm x 2.1 mm I.D.) at 35 ° C with the following conditions: 10:90% acetate buffer (pH 5.5-40 mM): acetonitrile as eluent and wavelength of detection 254 nm. The proposed HILIC method exhibited high precision (RSD% < 0.5%), concentration ranges of 100-5500 ppb, and the lowest detection limit was 6.8167 ppb with a coefficient of determination of 0.9998 for cefotaxime. The findings of the method were used statistical tests compared to the British Pharmacopoeia protocol, which did not show any difference in accuracy among the methods