The Rwenzori region in Uganda, a global biodiversity hotspot, is currently undergoing exponential economic and population growth, which puts continuous stress on its freshwater ecosystems. In Sub-Saharan Africa, biomonitoring campaigns using region-specific biotic indices is limited, particularly in Uganda. In this research, we present the Rwenzori Score (RS), a new macroinvertebrate-based biotic index developed to specifically assess the aquatic health of Rwenzori streams and rivers. We collected and measured both biological and physicochemical variables and identified 34,202 macroinvertebrates, belonging to 64 different taxa. The RS was developed in two steps. First, using canonical ordination, we identified chemical variables that correlated significantly with gradients in macroinvertebrate assemblage distribution and diversity. Second, based on selected variables and weighted averages, we determined specific family indicator values and assigned pollution tolerance values (varying from 1: tolerant; to 10: sensitive) to a family. Finally, we established four water quality classes: poor, fair, good, and excellent. The RS is highly correlated with the Average Score Per Taxon System (p < 0.05), a well-known and widely used biotic index. The RS has 5 unique taxa that are not included in other regional indices. In this regard, the development of the RS is a beneficial tool for tailor-made biomonitoring that can contribute to the sustainable development of the Rwenzori stream and river basins.