Notoginseng total saponins (NGTS) extract has been frequently used for the treatment of cardiovascular disease and its complications in clinical practice. Several major saponins, such as notoginsenoside R₁ and ginsenosides Rg₁, Re, Rb₁, and Rd, are known, however, the minor ginsenosides that account for 25% of all the NGTS have yet to be elucidated. Herein, a step-wise multiple reaction monitoring (MRM)-based strategy employing staggered [M + HCOO]⁻ > [M − H]⁻ transitions was proposed for the homolog-focused detection of the minor ginsenosides in NGTS using liquid chromatography (LC) coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (LC-Q-Trap/MS), and the efficiency and accuracy of the established method were then assessed using LC coupled with hybrid ion trap/time-of-flight mass spectrometry (LC-IT-TOF/MS). The most important parameter for an MRM scan is the collision energy, which was tuned using an online optimization strategy to further advance the detection sensitivity, and the isotopic peaks shown in the MS² spectra assisted determination of the molecular weights of the unknown minor peaks. As a result of this MRM-based strategy, 107 minor ginsenosides were characterized by comparison with reference compounds and matching with mass cracking patterns, which was more efficient than a full scan analysis using LC-IT-TOF/MS. Moreover, an approximately 98% accuracy for the MRM-based profiling was finally confirmed by the target-list-dependent scan on LC-IT-TOF/MS. Taken together, these results suggest that MRM-based strategy could be used as a reliable tool for screening and identifying trace components in ginsenoside-enriched herbal products and other homolog-gathered extracts.