Major rivers in India are subject to ongoing impacts from urban drain discharges, most of which contain high levels of domestic and industrial wastewater and stormwater. The present study aimed to determine the levels of bioactive organic micropollutants at the discharge points of major urban drains compared to upstream and downstream sites. To achieve this, we employed a panel of in vitro bioanalytical tools to quantify estrogenic, androgenic, progestogenic, glucocorticoid and peroxisome proliferator-like activity in water extracts collected from two Indian cities in the Ganga Basin. Cytotoxicity of the water extracts in a human-derived cell line and the potential to cause oxidative stress in a fish cell line were also investigated. We found high levels of activity for all endpoints in samples directly receiving urban drain discharge and low levels at sites upstream from drain discharges. Estrogenicity was detected at levels equivalent to 10 ng/L 17β-estradiol, representing a high likelihood of biomarker effects in fish. Sites located downstream from drain discharges exhibited low to intermediate activity in all assays Effect-based screening tools such as those implemented here to provide a rapid and practical means of assessing the net potency of complex mixtures of contaminants that could potentially act as endocrine disruptors in aquatic biota through specific biological pathways. This approach can support the design of targeted chemical analyses, for example, by identifying priority classes of compounds for detection and monitoring. Using high-throughput screening tools could be a low-cost solution to monitoring micropollutants in the Ganga River as management approaches are implemented or may be applied in future studies with the greater temporal or spatial resolution to inform human and ecosystem risk assessments.