Hyperthyroidism is common in older cats, which necessitates frequent screening of serum total thyroxine (TT4) concentrations. Fast, cheap, and reliable methods to measure TT4 in cats are needed. The purpose of the study was the validation of a human TT4 enzyme immunoassay (EIA) for use with feline serum, and derivation of a TT4 reference interval (RI) for cats aged 9 years and older. Assay precision, reproducibility, and linearity were evaluated. Interference by hemolysis was also assessed. Method comparison studies between the human EIA and a previously validated radioimmunoassay (RIA) and chemiluminescent-enzyme immunoassay (CEIA) were performed. Healthy cats (> 9 years) were recruited from 3 UK first opinion practices. The human TT4 EIA demonstrated good precision and reproducibility, and adequate linearity. Hemolysis did not significantly alter measured TT4 concentrations until HGB > 8 g/L. Method comparison revealed proportional and constant errors between EIA and RIA/CEIA. The TT4 RI for cats (> 9 years) was calculated as 7.1-45.1 nmol/L (n = 49). The human TT4 EIA was successfully validated for use with feline serum and offers a rapid, cheap, and reliable method for determination of serum TT4 concentrations in cats.