Eukaryotic cells contain an extensive cytoplasmic network of actin filaments and microtubules that function as tracks for vesicle transport. The ability to visualise vesicle movement along both sets of filaments advanced rapidly when in vitro vesicle transport (motility) assays were developed. These assays utilise super-resolution, contrast-enhanced DIC microscopy to detect structures below the resolution of the light microscope including vesicles as small as 100 nm and microtubules 25 nm in diameter. Images are captured in real time and stored digitally for subsequent analysis. The standard motion analysis parameters include vesicle type, velocity, distances and path length. In vitro motility assays enabled the identification of important factors that are essential for the regulation of intracellular transport including motor proteins and their membrane receptors, the formation of hetero-motor complexes and transported cargoes.