A novel and simple hyphenated method was applied for the screening of alkaloids in S. flavescens. The approach combined easy sample extraction with an ultra-high-performance liquid chromatography (UPLC) and a high-resolution mass spectrometry (HRMS) in the data-dependent acquisition (DDA) method. With the optimized chromatographic program, ten alkaloids were screened. Among them, matrine, oxymatrine, oxysophoridine, sophocarpine, and oxysophocarpine were identified and quantified. The other five alkaloids were identified with accurate m/z value in MS1 and product ions in MS2. The quantification method was validated with specificity, linearity, recovery, and precision. The LOD and LOQ were determined. In twelve batches of collected S. flavescens roots, the identified ten alkaloids were all observed. The oxymatrine was the most abundant component, and sophocarpine was the least abundant in these samples. This comprehensive method is a powerful tool for the biochemical and clinical research of S. flavescens. It can also be adopted in resource utilization and related medicine quality control.