Objectives The objective of this study was to assess the absorption of transdermal ondansetron in healthy cats. Methods Five research cats with unremarkable complete blood count, biochemistry and urinalysis were used for both single- and multiple-dose application studies. For single-dose application, 4 mg ondansetron in 0.1 ml Lipoderm gel was applied once to the internal ear pinna. Blood samples were collected via jugular catheter over a 48 h period following administration (0, 15 mins, 30 mins, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h). For multiple-dose application, 4 mg ondansetron in 0.1 ml Lipoderm gel was applied for five consecutive days before blood samples were obtained in the same manner. Serum was separated and frozen prior to analysis. Ondansetron was measured via liquid chromatography coupled to tandem mass spectrometry. Results Analysis revealed no clinically relevant drug levels in serum after either single- or multiple-dose administration of 4 mg transdermal ondansetron. Conclusions and relevance Transdermal application of 4 mg ondansetron does not result in clinically relevant serum concentrations of drug. Despite characteristics of the drug that imply suitability for transdermal application, this does not appear to be an acceptable method of drug delivery for this medication at this dose. This study highlights the importance of assessing the suitability of each medication for transdermal administration.