Objectives The aim of this study was to evaluate performance of a feline-specific ELISA for the measurement of alpha-1-acid glycoprotein (AGP) and to establish a reference interval (RI). Methods Surplus serum samples with low (~200 µg/ml), medium (~450 µg/ml) and high (~745 and 930 µg/ml) AGP concentrations were used to assess the intra- and inter-assay coefficients of variation (CVs). The quality goal for bioanalytical method validation was a CV of <20%. Linearity was assessed by serial dilution of a sample with a high AGP concentration. Spike recovery was evaluated by mixing samples with low, medium and high AGP concentrations at different ratios. To establish the RI, residual serum samples from 51 healthy adult cats that were presented for health examinations or blood donation between August 2020 and June 2021 were included. Results The intra-assay CV was 8.5%, 4.3% and 4.0%, and the inter-assay CV was 18.8%, 15.5% and 11.5% for serum samples with low, medium and high AGP concentrations, respectively. Excellent linearity (R2 = 0.98) was demonstrated for AGP concentrations ranging between 251.6 and 954.4 µg/ml. Average recovery was 95.0–99.7%. The right-sided RI for AGP was 328 µg/ml (90% confidence interval 300–354). Age had a statistically significant impact (increasing values with older age, P = 0.0026), but sex did not ( P = 0.44), on AGP concentrations. Conclusions and relevance The ELISA was accurate and showed acceptable precision with the modification of dilution used in this study. AGP concentrations in this population appeared to increase with increasing age.