In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells. Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 min of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.