The accurate monitoring of surface cleanliness in terms of bacterial contamination is usually carried out using methods such as plate counts or replica plating. However these methods take at least eighteen hours to obtain results and do not determine the presence or amount of residual organic material on a surface, which may interfere with cleaning and disinfection. This work describes the application of fluorescent stains to cells (Listeria monocytogenes) and food soil (solubilized whey) to optimize a dual staining method that can be used in the quantitative analysis of surface cleanability. Seven different stains were tested at a range of concentrations (0.3%-0.001 mg/ml) and application methods. The best stain combination for differential staining of L. monocytogenes and whey food soil was 0.1 mg/ml rhodamine B with 0.1 g/ml DAPI. Differential staining of the cells and soil occurred regardless of the application method. This method has been successfully used to demonstrate the hygienic status of surfaces in an industrial situation. This novel work enables quantitative assessment of soils and cells on surfaces.