<p>Ensuring reliable tools for evaluating river ecosystem health is increasingly important in tropical regions where anthropogenic pressures and agricultural intensification threaten aquatic biodiversity. However, the use of reliable multimeric indices to assess river ecosystem health remains poorly developed in the tropical regions. This study developed and validated a benthic macroinvertebrate-based Index of Biotic Integrity (B-IBI) to evaluate river health in the Walawe River Basin, Sri Lanka. Benthic macroinvertebrate assemblages, together with physicochemical and habitat characteristics, were assessed across multiple stream sites representing reference and disturbed conditions during both dry and wet seasons. A total of 28 macroinvertebrate-based candidate metrics were selected for IBI development, and they were screened for their association with environmental variables using Pearson’s correlation, redundancy, and box-and-whisker analyses for discriminating reference and degraded sites. After the screening process, three candidate matrices were selected for final B-IBI development: Percentage Ephemeroptera + Plecoptera + Tricoptera (%EPT), Shannon diversity (H′), and the percentage of Crustacea and Mollusca. The final B-IBI, composed of key taxonomic and diversity-based metrics, effectively distinguished reference sites from degraded locations, demonstrating strong discriminatory power and robustness across varying land-use and environmental conditions. The developed index provides a regionally adapted and ecologically meaningful tool for river health assessment and contributes to advancing bioassessment frameworks for tropical river ecosystems.</p>