Tumour necrosis factor-alpha (TNF-α) production by malignant lymphoblasts has been identified in vitro and in vivo in mice and humans, respectively. The goals of this study were (1) to evaluate a novel single-sample TNF-α assay and (2) to determine whether TNF-α is increased in dogs with lymphoma prior to and following treatment. Canine TNF-α was analysed concurrently using the novel Siemens Immulite® single-sample automated ELISA and the previously validated Quantikine® standard ELISA. Serum from dogs with lymphoma and from breed-, age- and gender-matched control dogs was evaluated at two time points. Three of 25 (12%) dogs with lymphoma had detectable TNF-α at diagnosis, whereas none had detectable TNF-α following complete or partial remission. TNF-α was not detectable in control dogs. Despite 91% homology between human and canine TNF-α, the Immulite® automated ELISA failed to detect canine TNF-α. Serum TNF-α appears to have limited value as a tumour marker in dogs with lymphoma.