Cell signaling and endocytosis are intimately linked in eukaryotic cells. Signaling receptors at the cell surface enter the endocytic pathway and continue to activate downstream effectors in endosomal compartments. This spatiotemporal regulation of signal transduction provides opportunity for signal diversity and a cell-specific machinery of scaffolding/targeting proteins contributes to establish compartment-specific signaling complexes. Members of the annexin (Anx) protein family, in particular AnxA1, AnxA2, and AnxA6, appear to target their interaction partners to specific membrane microdomains to contribute to the formation of compartment-specific signaling platforms along the endocytic pathway. A major challenge to understand the impact of scaffolding/targeting proteins on spatiotemporal signal transduction along endocytic pathways is the identification, isolation, and functional analysis of low-abundance signal-transducing protein complexes in endocytic compartments. Here, we describe methods to isolate endosomes and to target signaling molecules to endosomes. Applying these methodologies to suitable animal or cell models will enable the dissection of signal transduction in the endocytic compartment in the presence or absence of annexins.