Human cardiac troponin I (cTnI) assays have been used in equine medicine, often without prior analytical validation for equine use. In the absence of appropriate validation, the clinical significance of assay results is uncertain and can lead to misdiagnosis. We followed the American Society for Veterinary Clinical Pathology guidelines and investigated linearity, precision, limit of quantification (LoQ), and comparative recovery for 6 commercial cTnI assays developed for use in human medicine. Clinically acceptable linearity was observed in assays A–D, whereas assay E did not detect equine cTnI in any sample. Comparative recovery revealed 1–3-fold differences between assay results, and low analyte recoveries (2.2–3.4%) were observed in assay F. Precision was investigated in assays A and B, and found to be within acceptable limits. The LoQ was 1.53 ng/L for assay A, and 0.031 µg/L for assay B. Assays A and B performed within clinically acceptable limits and were deemed suitable for use in equine medicine. Assays C and D did not undergo full validation but had acceptable linearity, which demonstrates their potential for use in equine medicine. Assays E and F are unsuitable for use in horses given issues with detection of equine cTnI. The variability in results between assays indicates that reference intervals and cutoffs for diagnostic decision-making are assay specific and should be established prior to adoption by diagnostic laboratories.