Vietnamese dandelion (Lactuca indica L.) is a medicinal plant used in traditional medicine for heat-clearing, detoxifying, and anti-inflammatory purposes. This study aimed to optimize an ultrasound-assisted extraction process to obtain an extract rich in luteolin and luteolin-7-O-glucuronide from L. indica leaves and to evaluate its in vitro anti-inflammatory activity. Response surface methodology with a Box–Behnken design was applied to investigate the effects of the solvent-to-material ratio, ethanol concentration, and extraction time on the contents of luteolin and luteolin-7-O-glucuronide, which were quantified by HPLC–DAD. The optimal extraction conditions were a solvent-to-material ratio of 30 mL/g, 60% (v/v) ethanol, and an extraction time of 30 min. The optimized extract exhibited marked anti-inflammatory activity in the red blood cell membrane stabilization assay, achieving 65,61 ± 2,46% stabilization, corresponding to 74,79 ± 2,8% relative to diclofenac sodium. These findings provide a scientific basis for optimizing and standardizing marker-oriented extraction of L. indica toward biologically active constituents.