The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single proteins in planar-supported biomimetic membranes. This chapter describes, in detail, the construction of a simple single-molecule microscope, which includes (1) a parts list, (2) temperature control, (3) an alignment procedure, (4) a calibration procedure, and (5) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar-supported bilayers on hydrophilically treated borosilicate and quartz. These include (1) POPC bilayers, (2) POPC/PEG-PE cushioned bilayers, (3) POPC/PEG-PE cushioned bilayers on BSA passivated substrates, and (4) a cushioned biomimetic membrane of the endoplasmic reticulum (ER). A procedure for the detergent-mediated incorporation of the transmembrane protein 5HT3A (a serotonin receptor) is also described. It can be a starting point for other large, nonself-inserting transmembrane proteins. A procedure for the detergent-free incorporation of cytochrome P450 reductase (CPR) and cytochrome P450 enzymes (P450) into an ER biomimetic is also described. The final experimental section of this chapter details different procedures for data analysis, including (1) quantitative analysis of mean squared displacements from individually tracked proteins, (2) gamma distribution analysis of diffusion coefficients from a small ensemble of individually tracked proteins, (3) average mean squared displacement analysis, (4) Gaussian analysis of step-size distributions, (5) Arrhenius analysis of temperature-dependent data, (6) the determination of equilibrium constants from a step-size distribution, and (7) a perspective associated with the interpretation of single particle tracking data.