The aerosol-OT (AOT)/water/isopropyl myristate microemulsion was investigated as a carrier in transdermal drug delivery of tetracaine hydrochloride. The study included in vivo analgesic studies on rats and histopathological, irritation, and oxidative stress measurements on mice. The tetracaine hydrochloride encapsulated in AOT/water/isopropyl myristate showed an eightfold enhancement in the analgesic response of drug compared to the aqueous solution of the drug as measured by the tail-flick method. The analgesic response of tetracaine hydrochloride, however, highly depended on the concentration of AOT and water of the microemulsion. The preliminary histopathological, irritation, and oxidative stress studies showed that AOT/water/isopropyl myristate microemulsion system is a safe transdermal carrier of tetracaine hydrochloride with a concentration of AOT in isopropyl myristate up to 21:79 w/w.