We describe a method for calculating rates of fluorochrome transport from unstirred layer (USL) concentration gradients measured using confocal microscopy. Isolated Malpighian tubules or guts of Drosophila melanogaster were secured to depression slides and bathed in saline containing a fluorescent compound. By measuring the concentration gradient of fluorescent organic anions (fluorescein, Texas Red) or the P-glycoprotein substrate daunorubicin in the USL adjacent to the epithelium we were able to calculate the transepithelial flux of the fluorochrome using Fick's equation. Dose–response curves for fluorescein and Texas Red based on USL concentration gradients near the surface of the Malpighian tubule were comparable to those based on collection and analysis of secreted fluid droplets. Rates of Texas Red and daunorubicin secretion were also calculated for the gut of second instar D. melanogaster larvae, a tissue that is too small for measurement of transport rates by other in vitro techniques such as cannulation and perfusion. Our results suggest that measurement of USL concentration gradients by confocal microscopy may be applicable to any fluorescent indicator of rapidly transported compounds.