We report on the use of diffusion measurements to gauge the fluidity and surface binding properties of a molecular monolayer. The monolayer film consists of octadecyl-l-phosphonic acid (ODPA) and controlled amounts of a lyso-phosphatidic acid tagged with the fluorescent probe BODIPY (BLPA). The monolayer films were formed using a Langmuir-Blodgett (LB) trough and deposited onto a glass slide. Monolayer morphology was characterized during film formation using Brewster angle microscopy (BAM). Fluorescence Recovery After Photobleaching (FRAP) microscopy was used to measure translational diffusion of BLPA and Fluorescence Anisotropy Decay Imaging (FADI) was used to measure rotational diffusion of the BLPA chromophore. These results provide information on the motional freedom of the probe and, importantly, on the strength of interaction between the probe and the support. Compositional variations in the monolayer give rise to changes in constituent dynamics that reflect intermolecular interactions. (C) 2016 Elsevier Inc. All rights reserved.