This protocol describes a single molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single molecule total internal reflection fluorescence microscopy, and allows probing single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single molecule fluorescence microscopy is used to probe the pulled down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. This ultra-sensitive assay requires at least 10-fold less reagents, is significantly faster and provides quantitative data compared to western blot analysis. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5 – 2.5 hours for data acquisition and analysis.