Intracellular cargoes are trafficked back and forth throughout the cytoplasm by kinesin and dynein motors that walk towards the opposite ends of microtubule filaments. It remains unclear how cargos are sorted and delivered to many destinations inside cells. Previously, we studied how minimally active motors can achieve unidirectional motility and generate force by performing high-resolution single molecule fluorescence and optical trapping assays in vitro. More recently, we turned our attention to activation, cargo binding and regulation of kinesin and dynein motors through in vitro reconstitution assays. We also use electron microscopy and fluorescence imaging to test whether cargos are sorted by signals that are encoded onto the microtubule tracks, either by microtubule-associated proteins or by tubulin post-translational modifications. I will also talk about our efforts to develop new fluorescence imaging methods to visualize how motors transport intracellular cargos back and forth along microtubules inside cells.