Cardiac troponin I (cTnI) is considered the gold standard biomarker for myocardial injury and shows a high degree of homology between humans and dogs. The ADVIA Centaur XP High-Sensitivity Troponin I (AC-cTnI-HS) assay has been validated for use in humans but not dogs. The study objectives were to analytically validate the AC-cTnI-HS assay in dogs, to assess correlation between the AC-cTnI-HS and a previous ADVIA Centaur TnI-Ultra (AC-cTnI-U) assay, to assess cTnI sample storage stability, and to clinically evaluate the AC-cTnI-HS assay in healthy dogs and dogs with cardiac disease. Canine serum samples were used for analytical validation. Intra- and inter-assay variability, dilutional parallelism, and spiking recovery were assessed. Samples from 196 client-owned dogs were evaluated (healthy dogs (n = 39) or dogs with congenital heart disease (n = 54), myxomatous mitral valve disease (n = 68), dilated cardiomyopathy (n = 15), or myocarditis (n = 20)). Inter- and intra-assay coefficient of variation (%CV) was between 2.8–41.4% and 3.8–30.2%, respectively, with pools with concentrations >20 pg/mL all having %CVs <10%. The observed to expected ratios for dilutional parallelism and spiking recovery experiments ranged between 92.3 and 266.7.0% and 84.3 and 108%, respectively. A strong correlation between the AC-cTnI-HS and AC-cTnI-U assays was observed (Spearman’s ρ = 0.927), though a proportional bias existed, with AC-cTnI-HS assay concentrations being proportionally lower than AC-cTnI-U assay concentrations. Serum samples stored at -80°C had stable cTnI measurements for up to 2.7 years and after a single freeze-thaw cycle. Healthy dogs and dogs with congenital heart disease had significantly lower cTnI concentrations than dogs in the other three groups. The AC-cTnI-HS assay precisely, reproducibly, and accurately measures cTnI concentrations in dog serum with cTnI concentrations >20 pg/mL.