Based upon ecological data provided by a 6-year study of native species assemblage structure and function in near-pristine Limahuli Stream (Kauai), The Hawaii Stream Index of Biological Integrity (HS-IBI) incorporates 11 metrics covering five ecological categories (taxonomic richness, sensitive species, reproductive capacity, trophic–habitat capacity, and tolerance capacity). The HS-IBI was shown to effectively distinguish stream biological condition on a continuum from undisturbed (near-pristine) to severely impaired in sampling of 39 sites (6 estuarine reaches) on 18 Hawaiian streams located on all major islands. A significant relationship was validated between relative levels of human impact occurring within-watersheds (determined through use of a landscape indicator) and IBI ratings with metrics responding predictably to gradients of human influence. For management interpretation of HS-IBI results, a framework comprised of five “integrity classes” (excellent–good–fair–poor–impaired) is provided which can be used to operationalize HS-IBI results obtained through standardized sampling of stream sites that “…translates into a verbal and visual portrait of biological condition.” Through its focus on native species, the HS-IBI incorporates evolutionary and biogeographic variation for the region with biological expectations based upon reference condition benchmarks established in near-pristine stream environments where ecological functioning is naturally self-sustaining and resilient to normal environmental variation. The methods and tools described in this study are appropriate for application in all perennial streams in Hawaii and may be adapted for use in streams on other tropical Pacific islands where native species assemblages persist in near-pristine stream environments.