A simple isocratic RP-HPLC stability indicating method has been developed and subsequently validated for the determination of Irinotecan HCl and its related substance (SN-38) in pharmaceutical dosage forms as per ICH guidelines. The separation achieved on a reversed phase Phenomenex Luna C18 Column (5µ, 250 × 4.60 mm) as a stationary phase and 0.5% trichloro acetic acid: Acetonitrile: Methanol (60: 20: 20 v/v/v) as mobile phase at a flow rate of 1.0 ml/min. The UV detection was performed at 372 nm.  The retention time for Irinotecan HCl and SN-38 was found to be 8.65 and 7.30 min respectively. The detector response was linear in the concentration range of 30-150 µg/ml. The respective linear regression equation being Y= 5233.x + 13299 with R2 = 0.999. The percentage of Irinotecan HCl in pharmaceutical dosage form was found to be 100.5% and the percentage of related substance (SN-38) in formulation was found to be 0.19%. The limit of detection and the limit of quantification were found to be 0.014 µg/ml and 0.045 µg/ml respectively. The results of the study showed that, the proposed RP-HPLC method was simple, rapid, precise, accurate and stability indicating, which can be used for the routine determination of Irinotecan HCl and its related substance (SN-38) in pharmaceutical dosage form.