Steady-state single molecule fluorescence anisotropy (SMFA) is described to quantify free and bound probe molecules from a Biotin-Neutravidin complexation reaction. By formulating a ratio of bound to the total number of molecules sampled (Nb/Nt ratio) we quantified the extent of binding. We report on a comparison of three methods to extract fluorescent bursts from single molecules from a ten-minute time trace. The impact on the N b/Nt ratio using either anisotropy values alone or anisotropy values combined with the difference in detector counts (Δn) were investigated. The data analysis methods reduced the random error due to scatter. Biotin-Rhodamine 110 (BR110) was used as the labeled probe for these studies. Neutravidin was used as the target protein. A competitive reaction between labeled BR110 probe and unlabeled Biotin was also investigated. The use of steady-state SMFA as a tool to probe molecular complexation will be useful in performing sensitive immunoassays, in drug discovery to investigate and enhance the binding of drugs to their substrates, and to study other molecular interactions.