Drug safety does not only depend on the active ingredient of the drug itself but also depends on the contamination contained in it. 1,2,3,9-Tetrahydro-9-methyl-3-methylene-4H-carbazol-4-one, or often called impurity D ondansetron is contamination in ondansetron which can endanger human health if the value is above the minimum (0.1 %) due to its toxicity. The qualitative and quantitative methods of selective and accurate for the impurity D ondansetron need to be developed to ensure the quality of the drug. Qualitative and quantitative analysis of drug contamination is often not possible due to it is constrained by the high-price of standard contaminant. The aim of this study is to obtain impurity D ondansetron that fulfills the requirements for characterization and purity as candidate for standard impurity; and obtain a method of analysis of impurity D ondansetron both qualitatively and quantitatively in ondansetron tablet which is valid in HPLC. The research experiments were carried out by hydrolyzing ondansetron in alkaline condition with optimum concentration 1 M NaOH for 30 h at 80 °C. Impurity D Ondansetron from degradation was successfully isolated by ethyl acetate as a solvent and hexane-ethyl acetate (6:4) as an eluent. The purity of Impurity D Ondansetron was measured using HPLC-DAD and obtained the purity index of 1.0000 and purity of 99.77 %. Impurity D Ondansetron was characterized using infrared spectroscopy, mass spectroscopic gas chromatography and HPLC-DAD.