A simple, sensitive, and specific gas chromatographic (GC) method was developed to determine the main bioactive sesquiterpene lactones, trilobolide-6-O-isobutyrates A and B (TBO-A and TBO-B), in Wedelia trilobata, a useful folk herb. A commercially available HP-5 capillary column (30 m x 0.25 mm i.d. x 0.25 microm) was utilized for the direct determination of TBO-A and TBO-B in W. trilobata. Calibration curves were obtained by spiking authentic compounds and the internal standard (ferulic acid) into W. trilobata samples before extraction. Extraction was carried out by refluxing the dried herb (0.5 g) for 1 h in methanol (25 mL). All calibration curves showed good linear regressions (r2 > 0.992) within test ranges. The assay was reproducible and accurate with the overall intraday and interday relative standard deviations and accuracies of less than 10% and more than 90%, respectively. The developed GC method was successfully utilized to analyze the TBO-A and TBO-B in aerial parts and flowers of W. trilobata, indicating that it was suitable for the quality control of this commonly used herb and related traditional Chinese medicines.