Cardiac troponin I (cTnI) is a cardiac-specific biomarker, used for the detection of myocardial injury. While rabbits develop naturally occurring cardiovascular disease, they are also an animal model for human disease; thus, rapid detection of cTnI has implications for both veterinary and human medicine. The objective of this study was to validate and establish a reference interval for a point-of-care (POC) cTnI assay in New Zealand White rabbits. In the first portion of the study, rabbit cardiac and skeletal muscle tissues were used to create homogenates, serially diluted with saline or rabbit whole blood, and run by repeated analysis on the POC assay. In the second portion of the study, a reference interval of peripheral whole blood cTnI was determined by robust methods from 49 New Zealand White rabbits. The least diluted cardiac muscle homogenates produced detectable cTnI (mean 23.12 ± 3.557 ng/mL), while skeletal muscle homogenates produced low to undetectable cTnI. The CV ranged from 0.00% to 32.51% for cTnI of diluted cardiac muscle homogenates. Rabbit cardiac homogenate diluted in blood had a linear relationship to cTnI concentration (Y = 0.2254 × X + 0.5396, R² = 0.975). The reference interval for cTnI in this population was less than 0.04 ng/mL. This POC assay may be useful when rapid detection of cTnI is needed and differentiation between normal and elevated values is required. Given the high CV, this assay may not be appropriate for cases that require high sensitivity or detection of low concentrations of cTnI.