Freshwater ecosystems are undergoing rapid thermal shifts, making it increasingly important to understand species-specific responses to these changes. Traditional techniques for determining a species’ thermal tolerance are often lethal and time consuming. Using the enzyme activity associated with the electron transport system (ETS; hereafter referred to as enzyme assay) may provide a non-lethal, rapid, and efficient alternative to traditional techniques for some species. We used largemouth bass Micropterus salmoides (Lacepede, 1802) to test the efficacy of using an enzyme assay to determine thermal tolerance and respiratory exploitation in response to variable acclimation temperatures. Three tissue types were dissected from fish acclimated to 20, 25, or 30 °C and used in ETS assays at temperatures ranging from 7.5 to 40 °C. While there were significant differences among tissue types and acclimation temperatures, maximal enzyme activity occurred from 25.23 to 31.91 °C. Fish lost equilibrium at 39–42 °C in traditional CT_max trials, significantly higher than the upper optimum range determined via enzyme assays. The ratio of enzyme activity to measured whole organism respiration rate decreased with increasing water temperature, with the largest changes occurring at the upper optimum thermal range determined by enzyme assays. Our results indicate that ETS analysis may prove useful for obtaining biologically relevant thermal tolerances.