To evaluate and quantify Irinotecan (R-enantiomer) / Irinotecan related compound D in Irinotecan hydrochloride trihydrate API, a high stereo-specific liquid chromatography technique was developed and validated. The partition was accomplished on ChiralpakIC-3 (150 x 4.6 mm 3µm) through a mobile fragment comprising 0.1 % v/v Formic acid in water and acetonitrile with 1mL/min, 250C, 20µL, 50C and 370 nmas flow rate, column temparture, injection volume, sample cooler temperature and detection wavelength. At 8.903 and 9.75 min, the retention time of Irinotecan (R-enantiomer) and Irinotecan (S-enantiomer) was determined. The resolution between Irinotecan (R-enantiomer) and Irinotecan (S-enantiomer) was found to be 2.4. The impurity acceptance limit is 0.2 %. The established method's precision, accuracy, sensitivity, linearity, specificity, and ruggedness were all verified in accordance with ICH recommendations. The qualifying sample LOQ was found to be 0.4 g/ml, while the minimal amount of sample needed for LOD detection was found to be 0.12 g/ml. The proposed reversed-phase method has been sophisticated and authenticated in accordance with ICH criteria and is capable of quantifying irinotecan enantiomer in irinotecan hydrochloride trihydrate API at trace level concentration. The specificity, linearity, and accuracy of the approach were used to guarantee its efficacy; as a result, it is appropriate for the task at hand, may be used successfully for routine laboratory analysis, and can be utilised for quality control.
 Keywords: Irinotecan, liquid chromatography technique, enantiomer impurity