We assessed water quality using physical, chemical, and biological characteristics in 37 streams between Puerto Maldonado and Cusco in Peru. Study sites ranged from ∼200 to 4000 m in elevation, with streams selected as pairs (control/natural vs. human impacted) along this gradient. In general, temperature decreased (R2 = 0.82) and other parameters increased [dissolved oxygen (R2 = 0.19), conductivity (R2 = 0.17), pH (R2 = 0.37)] with elevation. Macroinvertebrates were hand collected by professional entomologists and using leaf packs implemented by conservation workers. The professionals identified 213 unique taxa from the hand collections, with 80 species collected only one time. Data from control streams showed that as elevation increased, total richness (p = 0.008) and EPT richness (p = 0.050) decreased whereas Diptera richness increased (p = 0.002). NMS ordination indicated significant differences in macroinvertebrate assemblages when control streams from low and high elevations were contrasted. Hand collections also revealed significant differences between control and impact streams for total richness, EPT richness and % Diptera, but not for % EPT, % EPT richness, or % Diptera richness. The majority of the deployed leaf packs were successfully retrieved and contained many macroinvertebrates (Avg. = 141 individuals per pack). There were 98 unique taxa (family level or higher) identified from the leaf packs by the trained conservation workers. Fourteen of 15 macroinvertebrate community metrics (at the family level) were able to detect significant differences between control and impact sites. All of the family level metrics responded similarly across the elevation gradient except total richness, EPT richness, EPT count, and % Hydropsychids. Both the Costa Rica and the Virginia Save Our Stream Indexes were able to differentiate control from impacted streams using leaf pack data. Although the diversity of macroinvertebrates was higher for hand collections relative to leaf packs (due to greater habitats sampled and higher taxonomic resolution), leaf pack samples were better able to distinguish control from impacted streams. Specifically, leaf packs were able to discern impacts in streams at low elevation better than those at high elevation. Generally, macroinvertebrates indicated impact from urbanization to be worse than impacts from other human activities (i.e., gold mining and agriculture). Overall, hand collections will serve as an important reference of species diversity going forward while leaf packs processed by trained conservation workers are a viable method to monitor stream water quality in Peru and perhaps elsewhere.