Marine invertebrates such as sponges, cnidarians, bryozoans, and tunicates are considered to be prolific sources of novel biologically active natural products. Specimens from these organisms are composed of soft tissues with a high water and inorganic salt content compared to those from terrestrial counterparts. In addition, some of the secondary metabolites from these organisms tend to be more polar than those of terrestrial. These general characteristics have led to the establishment of differentiated protocols for metabolites derived from marine invertebrates. In this chapter, we describe an available protocol for the isolation of secondary metabolites from marine invertebrates, including extraction, fractionation, and purification procedures. While detailed protocols should be adapted to the characteristics of the specific organism being studied, the methods provided here can serve as an initial guide for designing those.