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The measurement of fecal cortisol/corticosterone metabolites (FCMs) is often used to quantify the stress response. The sampling method is relatively non-invasive, reduces concern for elevation of cortisol from the sampling method, and has been shown to measure cortisol more consistently without the daily diurnal rhythm observed in blood. Commercial ELISA (enzyme-linked immunoassay) kits offer benefits over previously validated immunoassay methods but lack validation. The objective of this study was to evaluate a commercial ELISA kit (Arbor Assays TM DetectX ® Cortisol ELISA kit, K003-H1, Ann Arbor, MI, USA) and provide analytical and biologic validation of equine fecal and plasma samples. Horses (4 male, 4 female, mean ± SD: 4 ± 5 yr) were transported for 15 min with limited physical and visual contact via a livestock trailer. Blood and fecal samples were collected pre- and post-transportation. Parallelism, accuracy, and precision tests were used to analytically validate this kit. Data were analyzed using PROC MIXED in SAS 9.4. Plasma cortisol concentrations increased in response to trailering (254.5 ± 26.4 nmol/L, 0 min post-transportation) compared to pre-transportation (142.8 ± 26.4 nmol/L). FCM concentrations increased 24 h post-trailering (10.8 ± 1.7 ng/g) when compared to pre-transportation (7.4 ± 1.7 ng/g). These data support that changes in FCMs can be observed 24 h post-stressor. In conclusion, the Arbor Assays TM DetectX ® Cortisol ELISA kit is a reliable, economic option for the measurement of biologically relevant changes in cortisol in equine plasma and FCMs.