Abstract: Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.