Molecular motor proteins are responsible for the long range transport of vesicles and organelles inside living cells. A small number of motor types transport thousands of distinct cargoes to various regions in the cell at the same time. This requires that intracellular transport be tightly regulated, yet the details of how motor regulators and cofactors tune motor function remain unknown in most cases. In-vitro studies at the single motor level have been instrumental in understanding the function of individual motors. In this thesis work I developed the methodology to extend in-vitro experiments to interrogate motor regulation at the single molecule level. I describe my modifications to the microscope setup as well as the acquisition cycle that made this possible. By combining differential interference contrast microscopy with single molecule fluorescence imaging and optical trapping I was able to manipulate and image the cargo while imaging a fluorescently-labeled regulator binding at the site of the motors. I used lipid droplets purified from Drosophila embryos as cargoes. Lipid droplets are carried by the opposite polarity microtubule motors kinesin and dynein in the embryos, and bind specifically to microtubules in-vitro. In the presence of ATP they exhibit long-range and short-range motility. For this proof-of-principle experiment I used fluorescently labeled AMPPNP, a non-hydrolysable analogue of ATP which binds to the motor domain of kinesin when microtubule-bound, to image the binding of the nucleotide to the motor and demonstrate the activity of the motors. While a large fraction of microtubule-bound droplets co-localized with a fluorescent AMPPNP molecule, non-specific binding of the nucleotide to the microscope slide surface prevented confirming the specificity of the colocalization events. Nevertheless, these data demonstrate the ability of the methodology to capture, in real time, the process of a regulator binding the motor at the single molecule level. / text