Rubia cordifolia is a common traditional Chinese medicine (TCM) used for centuries for the treatment of cough, inflammation of the joints, uterine hemorrhage, and uteritis. Mollugin is a major active component present in R. cordifolia and recognized as a potential anti-tumor compound. In this work, a microwave-assisted extraction (MAE) method has been developed for extracting mollugin from R. cordifolia. Several variables that can potentially affect the extraction yield, namely extracting solvent, microwave power, extraction time, and solid-liquid ratio were optimized. The separation and quantitative determination of mollugin was carried out by HPLC with UV detection at 254nm. Under appropriate MAE conditions, such as extraction time of 4min, ethanol concentration of 70% (v/v), microwave power of 460W, and solid-liquid ratio of 1:20 (g/mL), the extraction yield of mollugin from R. cordifolia with MAE was higher than conventional extraction methods such as Soxhlet extraction, heat reflux extraction, and ultrasonic-assisted extraction. Due to the considerable saving in time and its higher extraction yield, the proposed MAE procedure was obviously a more rapid and effective sample preparation technique.