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Growing interest in the commercialization of Thunbergia laurifolia leaves as an herbal tea and the use of its extracts as a nutraceutical has led to increased research surrounding its bioactive components and the standardization of the plant. Guided isolation through thin-layer chromatography (TLC) bioautography using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical as a detection reagent led to the isolation of a potent antioxidant compound, rosmarinic acid, from the leaves of T. laurifolia. This study is the first report of the isolation of rosmarinic acid from T. laurifolia. Rosmarinic acid showed significant DPPH-scavenging capacity with an EC50 value of 2.71 μg/ml. The high-performance liquid chromatography (HPLC) quantification of rosmarinic acid was accomplished. The developed method was applied to determine the amount of rosmarinic acid in T. laurifolia samples collected from different locations. The results demonstrated high variability in the amount of rosmarinic acid present in the samples, which correlated well with the observed antioxidant capacities detected by the freeradical scavenging assay. The HPLC method was also validated in terms of linearity, precision, and accuracy. The established HPLC method for the determination of rosmarinic acid, a bioactive principle, is a useful approach for standardization of the raw material and herbal preparations containing T. laurifolia.