In the present study, a system was developed for the qualitative and quantitative analysis of ginsenosides in the leaves of Panax notoginseng (PNL) using online comprehensive two-dimensional hydrophilic interaction chromatography and reversed-phase liquid chromatography coupled to a DAD detector and hybrid linear ion trap-Orbitrap mass spectrometry (HILIC×RP-DAD-ESI/HRMS/MSn). The system was configured based on the combination of an XBridge Amide column (150 mm × 2.1 mm, 2.5 μm) and Accucore Phenyl-Hexyl (50 mm × 4.6 mm, 2.6 μm) for the first and second dimensions, respectively. An additional water phase was optimized to dilute the eluent from the first dimension to decrease its elution strength in the second dimension. A back-flush trap column was selected as an interface to make up for the loss of efficiency and resolution due to the online dilution. Under the optimized conditions, a total of 235 ginsenosides were discovered from PNL, including 95 potentially novel ginsenosides, using the developed online HILIC×RP system. Moreover, the LC×LC-LTQ-MS system was validated for the quantitative analysis of 24 ginsenosides using the EIC contour plot, exhibiting satisfactory linearity (r≥0.9991). The online combination of HILIC×RP and HRMS system proved to be sufficiently powerful to provide unparalleled separation and identification of target analytes in samples with complex matrices.