The Thermo Scientific Dionex UltiMate 3000 LC system is applied for the assay and related substances analysis of Cefotaxime Sodium. The analysis was performed on a Thermo Scientific Syncronis aQ HPLC column using the method described in the JP XVI/KP X. The results obtained for resolution and symmetry factor met the criteria stated in the JP/KP. The results obtained for resolution between the peaks due to desacetyl Cefotaxime and Cefotaxime was 32.8(JP/KP criteria of minimum 20.0) and symmetry factor for cefotaxime was 1.0 (JP/KP criteria of maximum 2).