Two-dimensional liquid chromatography (2D-LC) separation systems, based on two independent columns with different separation mechanisms, have exhibited strong resolving power for complex samples. Therefore, in recent years, the exceptional resolution of 2D-LC has significantly advanced the chemical separation of natural products, such as complex herbs, greatly facilitating their qualitative and quantitative analysis. This paper aims to review the latest strategies of 2D-LC in the quantitative analysis of complex chemical compositions in natural products. To this end, the major advantages and disadvantages of various column couplings in 2D-LC are discussed based on specific studies, along with suggested solutions to address the identified drawbacks. Moreover, the applications of different detectors combined with the latest chemometrics in 2D-LC for accurate quantitative analysis of natural products are reviewed and discussed.