The role of tumor necrosis factor alpha (TNF-α), a cytokine shown to play a crucial role in human Crohn's disease patients, has not been documented in cats with chronic enteropathies. Also, currently, no validated assay for measurement of TNF-α in cats is available. The objective of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (ELISA) for the quantification of TNF-α in serum from cats. A sandwich ELISA was developed and analytically validated by assessment of detection limit, linearity, accuracy, precision, and reproducibility. A control range for serum fTNF-α concentration in healthy cats was established. In addition, serum concentrations of fTNF-α in 39 cats with chronic enteropathies were compared with those in 20 healthy cats. The detection limit of the assay was 38.4 ng/L. Observed-to-expected ratios for serial dilutions of 4 serum samples ranged from 75.1% to 111.9%. Observed-to-expected ratios for spiking recovery for 4 serum samples ranged from 91.3% to 129.7%. Coefficients of variation for intra- and inter-assay variability ranged from 3.9% to 7.6% and from 7.8% to 12.5%, respectively. The control range of the assay was < 38.4-223.5 ng/L. Serum concentrations of feline TNF-α were significantly higher in cats with chronic enteropathies and diarrhea than in cats with chronic enteropathies without diarrhea, or in healthy control cats. The ELISA described here was suitable for the quantification of fTNF-α in feline serum and should facilitate research into the importance of TNF-α in cats with chronic enteropathies.