Countercurrent chromatography techniques are increasingly used in the fractionation and purification steps of natural products due to their excellent versatility and efficiency. However, one of the major bottlenecks in liquid-liquid chromatography is the solvent system selection. In this work, a NMR-based analysis of complex solvent mixtures was developed for biphasic system selection and modification. This allowed us to develop more versatile separation conditions, such as gradients. Additionally, we hyphenated our countercurrent chromatography device with NMR and mass spectrometry.