Abstract: OBJECTIVE To elucidate the critical quality attributes of Shaoyao Gancao decoction(SGD) in the treatment of spasmodic diseases based on serum pharmacochemistry, network pharmacology and isolated intestinal smooth muscle contraction experiments. METHODS The blood migration components in SGD were analyzed and identified by UHplC-Q-Exactive Orbitrap MS. Cytoscape software was used to construct the network diagram of "blood components- spasmolytic target-pathway" and screen the core functional components of SGD in spasmolytic. The core components were verified by isolated intestinal smooth muscle test, and the critical quality attributes of SGD spasmolytic efficacy were clarified. RESULTS According to the accurate molecular weight and fragment ion information provided by high resolution mass spectrometry, combined with standard substance comparison and relevant literature reports, a total of 17 SGD prototype components were identified, included oxypaeoniflorin, albiflorin, paeoniflorin, isoliquiritin, liquiritin, glycyrrhizic acid, benzoylpaeoniflorin, licochalcone B, ononin, liquiritigenin, iso liquiritigenin, licochalcone A, licochalcone C, glabridin, glycyrrhetinic acid, gallic acid. Network pharmacology predicted that the key SGD antispasmodic active ingredients of glabridin, liquiritigenin, albiflorin, paeoniflorin and benzoylpaeoniflorin may exert antispasmodic effects through GRIN2A, VCP, ABCB1, CYP2C9, CYP3A4, CHRNB2, SNCA and other targets, acting on drug metabolism-cytochrome P450, neuroactive ligand- receptor interactions, cAMP, calcium signaling pathway, etc. Verification tests showed that the glabridin, liquiritigenin, albiflorin, paeoniflorin and benzoylpaeoniflorin could reduce the maximum contractile evoked by acetylchloine, and albiflorin and paeoniflorin exerted significant spasmolytic effects with the inhibitory response for acetylcholine evoked contraction(P<0.05 or P<0.01). CONCLUSION The critical quality attributes of SGD antispasmolysis efficacy are determined by establishing the screening method of the classic decoction standard, which provide experimental basis for the accurate quality control of SGD and the secondary development of SGD products.