Tricaine methanesulfonate (MS-222) is a bath anesthetic agent commonly used in amphibians, but few studies have demonstrated its efficacy in a wide variety of anuran species. In this study, White's tree frogs (Litoria caerulea; n = 9) were used in a blinded, randomized, complete cross-over study to assess the anesthetic effects of two MS-222 concentrations (0.5 g/L and 2 g/L). Frogs were placed in MS-222 to induce chemical restraint. Heart and gular rates and palpebral, corneal, withdrawal, and righting reflexes were measured every 5 min. Frogs were removed from the anesthetic solution when reflexes were lost or after 25 min. Only mild sedation was induced with 0.5 g/L MS-222 after 25 min in all frogs, and surgical anesthesia was induced in all frogs with 2 g/L MS-222 within 5–20 min. Time from rinsing with distilled water to regaining reflexes in the 2 g/L group ranged from 10 to 43 min. There was a time-dependent decrease in heart rate with no significant difference between treatments. There was a significant decrease in gular rate for the 2 g/L dose compared to the 0.5 g/L dose. These results suggest that 0.5 g/L MS-222 can be used for mild sedation to facilitate diagnostic techniques, and 2 g/L MS-222 can be used to induce surgical anesthesia in White's tree frogs.