The kidney is highly metabolically active, and injury induces changes in metabolism that can impact repair and fibrosis progression. Changes in the expression of metabolism-related genes and proteins provide valuable data, but functional metabolic assays are critical to confirm changes in metabolic activity. Stable isotope metabolomics is the gold standard, but these involve considerable cost and specialized expertise. Both the Seahorse bioflux assays and substrate oxidation assays in tissues ex vivo are two relatively cost-effective assays for interrogating metabolism. Many institutions have access to Seahorse bioflux analyzers, which can easily and quickly generate data, but guidelines to enhance reproducibility are lacking. We investigate how variables (e.g. primary vs. immortalized cells, time in culture) impact the data generated by Seahorse bioflux analyzers. In addition, we show the utility of (3)H-palmitate, a new approach for assessing fatty acid oxidation (FAO) in the kidney, in uninjured and injured kidney cortices. The (3)H-palmitate substrate oxidation assays also demonstrate significant sex-dependent and strain-dependent differences in rates of fatty acid oxidation. These data should facilitate metabolic interrogation in the kidney field with enhanced reproducibility.NEW & NOTEWORTHY We show significant metabolic differences between both primary and immortalized cells, and among primary cells with different durations in cell culture. In addition, (3)H-palmitate oxidation in tissue ex vivo is described as a method novel to the kidney for assessing the complete oxidation of long-chain fatty acids. This method shows that female mice have significantly increased fatty acid oxidation across two different strains of mice and significant strain-specific effects on metabolism.