Here we present a detailed guide for performing in vitro trafficking assays. These are high-resolution light microscopy assays designed to look at the cytoskeletal filament-based trafficking of cellular organelles. The strategy is to partially purify organelles from lysed mammalian cells and freeze them as single-use aliquots. The organelles are then thawed and allowed to bind microtubule and actin filaments that have been coated onto handmade optical microchambers. Time lapse multichannel fluorescence microscopy is then performed to identify specific vesicles and associated proteins and to observe and quantify how the material is transported. These protocols were initially developed to study rodent liver endosomes but are adapted here for the study of cultured cells and include commentary on their use with other types of organelles.