In the past decade, liquid chromatography-mass spectrometry (LC-MS/MS) has become pivotal in clinical diagnosis, drug discovery, and bioanalytical science due to its high sensitivity and rapid analysis. We have developed an ultrasensitive and robust LC-MS/MS method for the simultaneous detection and quantification of cyclosporine A (CsA) and urolithin A (UA) employing ascomycin (ASC) and naringenin (NAR) as internal standards (ISTDs). The method was validated for clinical use, revealing interspecies differences between human plasma and other mammals (e.g., mouse, rat, feline, canine, and bovine serum). Validation parameters, including accuracy, precision, limits of quantification, specificity, selectivity, carryover, linearity, stability, and recovery, met acceptable ICH standards. Linear regression across the full calibration range (1-250 ng/mL for CsA and 0.5-125 ng/mL for UA) yielded an average R