Here we describe a method for imaging the position of nanoparticles within a 3D tissue-engineered model using confocal laser scanning microscopy (CLSM). The ability to track diffusion of nanoparticles in vitro is an important part of trans-dermal and trans-mucosal drug delivery development as well as for intra-epithelial drug delivery. Using 3D tissue-engineered models enables us to image diffusion in vitro in a physiologically relevant way; not possible in two-dimensional monolayer cultures (MacNeil, Nature 445:874-880, 2007; Hearnden et al., Pharmaceutical Res. 26(7):1718-1728, 2009). CLSM enables imaging of viable in vitro models in three dimensions with good spatial and axial resolution (Georgakoudi et al., Tissue Eng 14:1-20, 2008; Schenke-Layland et al., Adv. Drug Del. Rev. 58:878-896, 2006). Here we show that fluorescently labelled nanoparticles can be visualised, quantified, and their position within the cell can be determined using CLSM.