A Fluorescence Recovery after Photobleaching (FRAP) method to study protein diffusion on solid surfaces was developed using confocal microscopy. By photobleaching a rectangular area of the sample, fluorescence recovery could be analyzed as one-dimensional diffusion, leading to s simplified mathematical expressions for fitting the data. The method was tested by measuring bovine serum albumin (BSA) diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. Furthermore, the method was used to analyze the diffusion of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). Using ellipsometry data of TLL adsorption, fluorescence intensity could be calibrated to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient was lowest on the OTS-surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important