Introduction The Chinese medicine formulation, tumour‐shrinking decoction (TSD, FM1523), which consists of 15 natural medicines, is used for uterine fibroids (UFs) therapy and possesses excellent clinical therapeutic effect. Objective To develop a sensitive and validated analytical method for the simultaneous quantification of four crucial bioactive compounds including isorhamnetin‐3‐O‐neohesperidoside, curcumin, peimine and tetrahydropalmatine in the principal formulation of this decoction. Methods An ultra‐performance liquid chromatography coupled tandem mass spectrometry (UPLC‐MS/MS) with an electrospray ionisation (ESI) source in multiple reaction monitoring (MRM) mode was conducted to investigate these bioactive compounds in the TSD. The chromatographic separation was performed on a C18 column when the flow rate was adjusted at 0.2 mL/min with gradient elution of acetonitrile–water with 0.1% formic acid. Accelerated solvent extraction (ASE) method with higher extraction efficiency was employed for TSD sample pre‐treatment. Results The linearity, limit of detection (LOD) and limit of quantification (LOQ) were determined for this analytical method. The mean recoveries of the compounds were determined between 100.23% and 104.02% with satisfactory relative standard deviation (RSD) in the ranges of 2.65% to 3.81%. The precision was evaluated by intra‐day and inter‐day tests, which revealed RSD within the ranges of 1.21% to 2.14% and 1.24% to 2.32%, respectively. Conclusion The bioactive compounds of TSD samples were successfully quantified via UPLC‐MS/MS with MRM mode. This study could help to evaluate the pharmacokinetic study of TSD during clinical applications and present a facile strategy for quantifying bioactive compounds in traditional Chinese Medicine decoction.