The purpose was to determine the safety and achievable serum concentrations of transdermally administered phenobarbital in healthy cats. The hypothesis was that transdermal phenobarbital would achieve therapeutic serum concentrations (15–45 µg/mL) with minimal short-term adverse effects. Enrolled cats had normal physical and neurologic exams and unremarkable bloodwork. Transdermal phenobarbital in a pluronic lecithin organogel–based vehicle was administered at a dosage of 3.0–3.1 mg/kg per ear pinna (total of 6.0–6.2 mg/kg) every 12 hr for 14 days. Serum phenobarbital concentrations were measured 3–6 hr after dosing at seven different times over 15 days. The mean and median serum concentration of phenobarbital at study completion were 5.57 and 4.08 µg/mL, respectively. Mean peak concentration and mean time to peak concentration were 5.94 µg/mL and 13.3 days, respectively. Mild adverse effects were observed. Potency was analyzed in three replicates of the transdermal phenobarbital gel administered; potencies ranged from 62.98 to 82.02%. Transdermal application of phenobarbital in healthy cats achieves a detectable, but subtherapeutic, serum concentration and appears safe in the short term. The use of therapeutic drug monitoring is recommended when this formulation of phenobarbital is used to ensure therapeutic serum concentrations are achieved.