L-theanine has been evaluated in several clinical trials, demonstrating beneficial effects in dogs and cats in recent years. However, specific safety trials of L-theanine in these target species in the public domain are lacking. The present study was designed to evaluate the safety of L-theanine at the recommended dose, and to demonstrate an adequate margin of safety in dogs. Beagle dogs (4 per sex/group; 9.54-14.53 kg) received one or five hard chews orally per day for 28 days; four control dogs were not given any chews. Each 25-g hard chew contained 0.184% L-theanine. Blood was collected on Days 14, 15, and 28. Body weights and clinical examinations were recorded weekly, and animals were returned to stock at study completion. All animals ate the chews spontaneously within one hour, demonstrating good palatability; there was no effect on food consumption. No abnormalities were noted during the clinical observations throughout the study. Mild or moderate symptoms were recorded in some animals, including development of otitis in one animal and raised body temperature in two females. However, all symptoms were determined not to be treatment-related and to be within normal physiological range. No treatment-related changes in body weight were observed. While diarrhea was recorded in a majority of the animals, including once in the controls, these effects were also present during the acclimation period, in general, for one dog in each housed pair and were determined not to be dose-related. Some individual hematology values fell outside the physiological ranges. For example, a few individual white-blood-cell counts were low in control-group animals on Day 14 or elevated in high-dose-group dogs before treatment. However, all mean hematology values were within the respective normal physiological ranges. Starting at Day 14, some animals in all groups had globulin values just below the reference range (mean 22 g/L, compared to 25-45 g/L); on Day 28, only the lower dose group remained lower (24 g/L). Similarly, mean chlorine values were just over the reference range of 109-122 mmol/L for all groups, and only remained elevated in the 5x dose group on Day 28 (123 mmol/L). Thus, while some minor differences in pre- and post-treatment serum chemistry results were observed, they were determined not to be clinically significant. In conclusion, the L-theanine product was found to not cause adverse effects when administered to dogs orally for 28 days, including up to the targeted margin of safety of 5x the recommended dose.