Ethylene glycol (EG) toxicity is commonly encountered in dogs and cats. The purpose of the current study was to determine if the Catachem test kit (Catachem Inc., Oxford, Connecticut) could precisely and accurately detect the presence of EG added to serum and plasma from 6 dogs and 4 cats. Serum and plasma samples were spiked at various concentrations of EG (0, 20, 60, and 100 mg/dl) and analyzed using the Catachem kit. Twenty randomly selected samples were also submitted for gas chromatography-mass spectroscopy (GC-MS) analysis of EG concentration, which was considered the gold standard. Inter- and intra-assay coefficients of variation (CVs) were calculated. Bland-Altman analysis was performed to compare the Catachem results to the GC-MS analyses. Analysis of serum samples showed a bias of 8.48 mg/dl (95% limits of agreement: 17.8 to -0.9 mg/dl) while spiked plasma samples had a bias of 7.32 mg/dl (18.1 to -3.5 mg/dl). Intra-assay CV was 0.7%. Interassay CV ranged from 1.2% to 2.0%. For all samples, the Catachem kit read higher than GC-MS values and slightly overestimated in vitro concentrations. The Catachem test kit is an accurate quantitative test for EG in dogs and cats that may aid in timely recognition of EG exposure. Because of the positive bias in all samples, some pets may receive treatment unnecessarily. However, animals with blood EG concentrations at or above the published lethal serum or plasma concentration will be readily identified so that treatment may be initiated.