The single molecule pull-down (SiMPull) assay allows for capture of individual proteins or macromolecular complexes that can then be interrogated via single molecule imaging. We describe several technical improvements over previous protocols that make it possible to directly detect and quantify the phosphorylation state of thousands of individual membrane receptors, and thereby estimate both the fraction of receptors phosphorylated at specific tyrosine residues and the frequency of multisite phosphorylation. These improvements include 1) the reduction of autofluorescence in the green spectral channel; 2) a simplified imaging chamber that accommodates higher sample number with lower sample volume; 3) corrections for membrane receptor surface expression; 4) three-color multiplex imaging; and 5) corrections for steric hindrance of dual antibody binding. These improvements enabled the first direct detection of multisite phosphorylation on full-length Epidermal Growth Factor Receptor (EGFR) and revealed that phosphorylation fraction varied by tyrosine residue. These SiMPull measurements provide a new level of detail in the status of receptor phosphorylation that was previously inaccessible with traditional biochemical techniques.