Chromatography has undergone significant evolution as a robust separation technique, adapting to meet the demands of various scientific and industrial applications. This review delves into both traditional and modern chromatographic methods, exploring their foundational principles, recent advancements, and applications across fields like pharmaceuticals, environmental monitoring, and food safety. Modern technologies like gas chromatography-mass spectrometry (GC-MS), two-dimensional liquid chromatography (2D-LC), and ultra-high-performance liquid chromatography (UHPLC) have significantly increased the accuracy of speed and sensitivity of analytical processes, allowing for complex sample examination with greater precision. Chromatography use in natural product research is investigated., along with emerging trends like eco-friendly chromatography and portable miniaturized systems. Additionally, this review provides a comparative analysis of key chromatographic techniques—such as gas chromatography, thin-layer chromatography, and high-performance liquid chromatography —to direct method selection according to analytical goals. Finally, future perspectives are discussed, such as the integration of Machine learning and artificial intelligence in data interpretation, which promises to further expand the versatility and applications of chromatography.