To investigate the effect of second messenger pathways on the uterine smooth muscle contraction and their associated mechanisms, and compare the evaluation methods. Preparation of uterine smooth muscle strips from healthy pregnant 18-21 d SD and non-pregnant rats. When the contraction of muscle strips was stable, we conducted gradient administration: PDE4 inhibitors (Z90), prostaglandin PGE2, adenylate cyclase inhibitor (SQ 22,530), cAMP analogs (dbcAMP) and AMPK agonists (AICAR), solvent dimethyl sulfoxide (DMSO) as controlled. Gradient administration of acetylcholine (Ach) and oxytocin (oxytocin) induced the contraction of muscle strips. The tension transducer and biological information collecting system were applied to record the changes, including duration, dilation tension, contraction tension, peak height, and mean tension, before and after different administration. Principal components analysis was adopted to evaluate the five changes. SQ 22,530, DMSO, cAMP alone had no significant effect on the contraction of uterine smooth muscle; Z90 can inhibit the spontaneous contraction of pregnant uterine smooth muscle strips; dbcAMP and AICAR can antagonize acetylcholine and oxytocin-induced the contraction of pregnant uterine smooth muscle strips. Z90, SQ 22,530 + Z90, dbcAMP, AICAR can inhibit the uterine contraction peak, diastolic amplitude, average muscle tone and contraction duration of the pregnant uterine smooth muscle in a concentration-dependent manners. At the same time, we compared the parameters, which reflect the contraction of uterine smooth muscle, and conduct main components analysis to determine the effect of the drugs. The second messenger cAMP and its related components ATP, 5'- AMP, AC, PDE, PKA, and AMPK can affect the uterine smooth muscle contraction via related signaling pathway in rats, and principal components analysis can be adopted to evaluate the smooth muscle relaxant.