A simple, rapid, accurate, robust, sensitive, and stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) analytical method has been developed for the separations of related impurities and degradants of topotecan in topotecan injection. Forced degradation studies performed on topotecan injection formulation revealed that the compound (topotecan) is extremely susceptible to all hydrolytic, oxidation, light exposure, and thermal stress. The method was tested in the presence of commonly used formulation excipients such as mannitol and tartaric acid. Separation of topotecan from its degradation products and three related impurities (Imp-A, Imp-B, Imp-C) as process impurities was achieved on ACE® C18, 150 × 4.6 mm i.d, 3 µm, stainless steel analytical column using a gradient elution. The elution of impurities employed multi-step gradient programmed mobile phase consisting of 0.02 M ammonium acetate (pH, 4.2) as mobile phase-A and a mixture of methanol and isopropyl alcohol in the ratio of 750:250 (v/v), respectively, as mobile phase-B. The mobile phase flow rate was maintained at 0.8 mL min−1. The column eluent was monitored at 267 nm. The column oven temperature was 25°C. The same method was also extended to LC-MS with the same gradient conditions used to identify the degradation products observed under forced degradation studies. The method was validated according to FDA, USP (general chapter 1225), and ICH guidelines, and statistical analysis of the data proved to be suitable for stability testing at quality control. The linearity of the proposed method for all the aforementioned related impurities and topotecan was investigated in the range of LOQ to 4.9 µg mL−1, respectively. The method was shown to separate topotecan well from the degradants and commonly used excipients. The specificity of the method was established through peak purity testing using a photodiode array detector.