Isolation of compounds in a pure state from natural sources is the most important, yet can be a difficult and time-consuming, step in natural product research. It begins with the process of extraction followed by various separation techniques. One such separation technique is the solvent partitioning method, which usually involves the use of two immiscible solvents in a separating funnel. In this method, compounds are distributed in two solvents according to their different partition coefficients. This technique is highly effective as the first step of the fairly large-scale separation of compounds from crude natural product extracts. In this chapter, the generic protocols of this separation technique in the purification of natural compounds, especially phytochemicals of relatively small molecular weights, i.e., in the range of 100–1500 Da, have been outlined.