DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure-functional analyses and biochemical reconstitutions. This limitation can be overcome by single-molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes "single-molecule sorting", a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post-translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification.