Quaternary ammonium salts (QAs) are a class of highly active compounds widely used in medicine and agriculture. However, many QAs lack a conjugated system, making their recognition and quantitation challenging. Stachydrine is a representative unconjugated QA with a high content in Leonurus japonicus Houtt. (Lamiaceae) and serves as a quality control compound for the aerial parts of L. japonicus (LJH). Based on previous studies of LJH and biosynthetic principles of plants, we speculated that a series of undiscovered stachydrine analogues (STAs) may be present in LJH. Using HILIC-QTOF MS technology, this study successfully identified other four STAs besides stachydrine. To further determine the content of total STAs, a quantitative 1H NMR (qHNMR) method was established. Notably, the effects of different concentrations of acids and bases on chemical shifts (δH) and the quantitation accuracy of STAs were investigated. Finally, the total STAs in LJH from different origins were quantified by the optimized qHNMR method, and it was found that the content of total STAs in LJH from Zhejiang province was higher than that from other provinces. The comprehensive use of HILIC-QTOF MS and qHNMR in this study provides a reference for the rapid recognition and comprehensive quantitation of unconjugated QAs.