Technical Abstract: We developed and validated a whole-body cortisol extraction technique for catfish fry. Their small size (< 1 g) makes it difficult to measure cortisol, a common indicator of a stress response, using conventional assay methods. Three volume enhancement methods were tested: CAL method (zero calibrator A buffer added to lipid extract), PBS method (phosphate buffered saline added to lipid extract), and VO method (food grade vegetable oil added to lipid extract). The volume enhancement extracts were evaluated using a commercial radioimmunoassay kit. Sensitivity, accuracy, precision, reproducibility, and parallelism could not be determined for the PBS method as cortisol levels were not detected in any of the extracted samples. Diluted extract samples were parallel to the stand curve for the CAL and VO methods. Sensitivity, accuracy, and recovery of diluted samples were better for the CAL method compared to the VO method. Intra-assay coefficient of variation (CV) for the CAL and VO methods were 7.3% and 8.3%, respectively while inter-assay CV were 9.6% and 10.6%, respectively. Based on the sensitivity, accuracy, precision, reproducibility, and parallelism results, we conclude that the CAL method is the most appropriate method for volume enhancement of catfish fry lipid extract. Using the CAL method to detect cortisol in catfish fry, fish were stressed daily for 2 wks. Fry weights were similar between stressed and control fish throughout the study while whole-body cortisol levels were higher (P < 0.01) in stressed fish after one day of stress. These data show the CAL method can effectively measure whole-body cortisol in catfish fry.