In principle, the isolation of secondary metabolites from microbes does not differ from their isolation from other organisms. The extraction procedure may of course be quite different, especially if it is carried out in an industrial scale, but when an extract containing the metabolites of interest is at hand, it is the same palette of adsorbents and chromatographic techniques that provide the major tools for the fractionation and eventual isolation of the pure compounds. Compared to plants, in which one is sure to find secondary metabolites of certain types, e.g., flavonoids, microbes can be expected to produce virtually anything and it is important to go about the fractionation procedure with an open mind. This chapter presents an overview of preparation of extracts from microbial sources, and various methods and strategies involved in the isolation and characterization of microbial natural products.