Background: Cardiac troponins are gold-standard biomarkers of myocardial injury. There is a need for validation of assays with higher availability and lower costs in veterinary medicine. Objectives: The primary aim of the present study was to perform an analytical validation of the IMMULITE 2000 TnI assay for use in dogs and cats. A secondary aim was to evaluate its agreement with the previously validated and sensitive Siemens ADVIA Centaur TnI-Ultra assay. Methods: Intra- and inter-assay variation, detection limits, the linearity under dilution, and a sample addition study (modified spike-and-recovery analysis) were investigated to assess analytical performance in 15 canine and 15 feline serum samples. Agreement between the assays was evaluated by correlation and Bland-Altman analyses including an additional 99 canine serum samples. Results: Intra-assay variation of cTnI in canine and feline serum was 3.71% and 4.68%, while inter-assay variation was 5.88% and 6.54%, respectively. The assay performed with acceptable linearity within a clinically relevant range of serum cTnI concentrations. The sample addition study revealed insufficient recovery in the range of 71.9%-81.4% for dogs and 62.6%-75.7% for cats. This was considered to be due to a negative matrix effect. A significant correlation between the assays was found, and the Bland-Altman analysis showed acceptable agreement for a wide range of concentrations, but revealed a proportional error, with the IMMULITE TnI assay consistently measuring a higher concentration than the Centaur TnI-Ultra assay. This was relevant only at high serum cTnI concentrations. Conclusions: The IMMULITE TnI assay is considered acceptable for clinical use in dogs and cats.