We have used coherent anti-Stokes Raman scattering (CARS) microscopy as a novel and rapid, label-free and non-destructive imaging method to gain structural insights into live intestinal epithelial cell cultures used for drug permeability testing. Specifically we have imaged live Caco-2 cells in (bio)pharmaceutically relevant conditions grown on membrane inserts. Imaging conditions were optimized, including evaluation of suitable membrane materials and media solutions, as well as tolerable laser powers for non-destructive imaging of the live cells. Lipid structures, in particular lipid droplets, were imaged within the cells on the insert membranes. The size of the individual lipid droplets increased substantially over the 21-day culturing period up to approximately 10% of the volume of the cross section of individual cells. Variation in lipid content has important implications for intestinal drug permeation testing during drug development but has received limited attention to date due to a lack of suitable analytical techniques. CARS microscopy was shown to be well suited for such analysis with the potential for in situ imaging of the same individual cell-cultures that are used for permeation studies. Overall, the method may be used to provide important information about cell monolayer structure to better understand drug permeation results.