During decoction process, the ingredients of herbal formula interact with each other, such that therapeutic properties and chemical extraction characteristics are altered. The crude drugs, Cinnamomum cassia (CC), Paeonia lactiflora (PL), and Glycyrrhiza uralensis (GU), are the main herbal constituents of Gyeji-tang, a traditional herbal formula. To evaluate the chemical interaction between CC, PL, and GU during the course of decoction, quantification of 16 marker compounds in the herbal decoction, performed using a Box-Behnken experimental design, was carried out by HPLC-diode array detection using validated method. Correlations between the amounts of marker compounds from CC, PL, and GU were assessed by multiple regression analysis. The results obtained showed that amounts of single herb marker compounds significantly changed (usually decreased) by decoction in the presence of other herbs and that these changes depended on the chemical natures of the markers and the herbal medicines present. Results also demonstrated that the extraction efficiencies of marker compounds increased when the proportion of the herb containing them was increased and decreased in proportion to amounts of herbs added. In conclusion, chemical interactions between compositional herbal medicines may occur when herbs are co-decocted. This study provides insight of understanding the herbal interactions in herbal formulae.