High performance liquid chromatography for estimation of Sulbactam Sodium and Ampicillin Trihydrate in their combine dosage form was developed and validated. The method was performed on Younglin Instrument  with Autochro-3000 Operation software using Varian C-18 (250 Χ 4.6 mm i.d, 5 μm particle size column and Ammonium acetate Buffer : Acetonitrile: Water (75:17:08, %v/v/v)  as mobile phase at ambient temperature. Detection was carried out at 228 nm in the concentration range 25-125 µg/ml for Sulbactam Sodium and 50-250 µg/ml for Ampicillin Trihydrate. The percentage recovery of Sulbactam Sodium and Ampicillin Trihydrate was found to be 99.15 -100.16 and 98.91-103.48 respectively.. Correlation coefficient for Sulbactam Sodium and Ampicillin Trihydrate was found 0.997 and 0.999 respectively. The Rt values for Sulbactam Sodium and Ampicillin Trihydrate were found to be 4.0 min ±0.02 and 5.97 min ±0.03 respectively. The method can successfully applicable to routine analysis.  And under Statistical analysis, Paired t-test is applied for comparison between developed method and reported method where, we reject the null hypothesis, because value of t is less than 0.05 so we can conclude that there is significance difference between the developed method and reported method for Sulbactam Sodium and Ampicillin Trihydrate.