The aim of this study was to compare canine and feline thyroxine (T4) results obtained by the Vcheck method with those obtained by the IMMULITE 2000 immunoassay, which had previously been validated for canine serum. The concentration of T4 in 53 canine and 44 feline serum samples was determined concurrently by the Vcheck and IMMULITE methods, the latter as the reference method. The T4 values were compared using the IMMULITE Youden plot and Deming regression analysis. According to the regression analysis, the two methods gave comparable results. Spearman’s correlation shows a very high consistency of the results obtained by the two analyzers. The results of the Vcheck method were comparable to those of the IMMULITE 2000 reference method, so Vcheck may be used as an alternative assay to evaluate the serum thyroxine concentration in dogs and cats for the diagnosis of thyroid gland disease.