Confocal laser scanning microscopy (CLSM) in combination with a fluorescently labeling enzyme dye, LavaPurple™, was demonstrated as a technique for the visualization of Thermomyces (Humicola) lanuginosa lipase (LIPHLL) and Candida antarctica lipase A (LIPCA) within a transparent latex coating. Addition of Teric Surfactants (C16 non-ionic Teric 475, 1.8% (w/w) or C10 non-ionic Teric 460, 2.0% (w/w)) significantly increased the accumulation of both LIPHLL and LIPCA to the surface of a latex coating. An α-naphthyl acetate substrate assay was used to quantify the accumulated lipase. The results derived from the acetate assay correlated with the enzyme accumulation (at the surface) observed in the CLSM images of the latex coating. This correlation demonstrated that the increased enzyme accumulation within the top 2 μm of the latex film was responsible for the increase in surface enzymatic activity. The combination of CLSM imagery and quantifiable image analysis provided a valuable tool for the optimization of surfactant concentrations for maximizing the activity of an enzyme (and potentially other additives) within a latex coating.