The pharmacological effects of Ophiopogon japonicus are influenced by its active substances like saponins and flavonoids, which are crucial for assessing its quality. An ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the qualitative and quantitative assessment of 20 saponins and flavonoids in O. japonicus. The average recovery of these compounds ranged from 70% to 130%, with relative standard deviations (RSD) between 0.11% and 16.46%. Planting O. japonicus in Santai County (Sichuan, China) and investigated the impact of 0.6, 1.3, 2.8, 5.6, and 11.3g/L concentrations of paclobutrazol on both root yield and the levels of saponins and flavonoids in O. japonicus. Lower paclobutrazol concentrations (1.3g/L) led to increased levels of saponins and flavonoids in O. japonicus such as ophiopogonin D′, ruscogenin and ophiopogonanone F, with paclobutrazol residues measuring 77.17μg/kg DW. This preliminary insight into the influence of paclobutrazol and production of saponin flavonoids in O. japonicus bears significance for its larger industrial production.