We present the Multi-metric Index of Biotic Integrity methodological approach that allows for the ranking of major river catchments based on pollution status in the Kenyan portion of Lake Victoria, Africa. The study has a broader applicability to all of Lake Victoria, other African Great Lakes, and all lakes that have riverine discharge. The method presented utilizes water quality and environmental data, local knowledge, and pre-existing literature. The parameters considered were sampled from 2016 to 2018 during the dry season (July sampling) and the wet season (March sampling). Separation power of Mann-Whitney U test (p < 0.05) qualified 11 discriminant metrics for both macroinvertebrate and fish samples into the scoring system of 1, 3 and 5 in the formulation of final Multi-metric Index of Biotic Integrity methodological approach. Rivers in the northern section had lower Multi-metric Index of Biotic Integrity methodological approach scores, as compared to southern counterparts. The Multi-metric Index of Biotic Integrity methodological approach ranking herein was validated by community perceptions on pollution levels. River Nzoia catchment emerged as the most polluted, followed by River Yala, River Kuja, and Sondu-Miriu. Siltation, domestic washing, litter and refuse emerged as the main agents of pollution. Management authorities ought to reinforce a balanced utilization of the vital water resources to minimize future impacts, and promote catchment wide practices that ensure ecological health sustainability of the lake ecosystem.