Background: This report aims to detail the use of the phosphorescence oxygen analyzer for in vitro investigation of thymic responses to pharmaceutical agents, in particular immunosuppressants and immunomodulators. Sirolimus (a highly specific inhibitor of the ‘molecular target of rapamycin’, mTOR) and ozanimod (an agonist of the sphingosine 1-phosphate receptor, recently approved for treatment of multiple sclerosis and ulcerative colitis) are used for this purpose. Methods: Thymic fragments from mice were placed in glass vials containing phosphate-buffered saline, bovine albumin, and Pd(II) meso-tetra (sulfophenyl) tetrabenzoporphyrin. The vials were sealed from air, and the cellular oxygen consumption was monitored as function of time. Results: The decline of dissolved oxygen concentration with time (d[O{}_2]/dt) was linear; thus, its rate (thymocyte respiration) was expressed as \muM O{}_2 min{}^{-1}. Cyanide inhibited respiration, confirming the oxygen consumption was in cytochrome oxidase. In age-matched mice, the rate of thymocyte respiration (mean \pm SD, in \muM O{}_2 min{}^{-1} mg{}^{-1}) was 0.046 \pm 0.011 (median = 0.043, range = 0.028 to 0.062, n = 10). In thymic fragments from littermates, this rate was inhibited in the presence of sirolimus (16% lower) or ozanimod (29% lower). Conclusions: Thymocyte respiration can serve as a surrogate biomarker for studying the mode-of-action and the cytotoxicity of immunotoxins and immunosuppressants.