© 2021 Bentham Science Publishers. Background: Over the last few decades, there has been a growing interest in Two-Dimensional Liquid Chromatography (2D-LC). This advanced technology has become a relevant solution for solving all the analytical challenges encountered in the pharmaceutical industry. The primary purpose of this review is to give a brief account of the developing techniques notable in 2D-LC. Methods: Review of recent literature on advanced separation techniques for complex mixtures revealed that 2D-LC is the most widely used technique in the pharmaceutical laboratory for the separation of complex samples. Among the various techniques used in 2D-LC, comprehensive 2D-LC and heart-cutting 2D-LC are highly used techniques. To enhance the resolution, further development has been made to heart-cutting 2D-LC called multiple heart-cutting 2D-LC, which has recently become a very popular technique in the pharmaceutical industry. Therefore research articles mentioning the use of multiple heart-cutting 2D-LC for separation of complex mixtures were reviewed. Results: The most crucial benefit of multiple heart-cutting 2D-LC is its reduced effort in method development and also the storage of multiple sample sections for further analysis. The multiple heart-cutting 2D-LC is suited, preferably for method development and impurity analysis of pharmaceutical substances, fine chemicals and offers better peak separation, which is otherwise not possible by comprehensive 2D-LC. Conclusion: The advantage of two-dimensional liquid chromatography over one-dimensional liquid chromatography is its high resolving power. Further improvement in resolution was achieved by using the advanced multiple heart-cutting 2D-LC which appears to have an imperative role in pharmaceutical and food analysis.