Unwinding of double-stranded DNA is required to create a single-stranded DNA template for essential DNA processes such as those involved in recombination, repair, and replication. A set of specialized enzymes called DNA helicases is dedicated to this purpose, catalyzing DNA strand separation by breaking hydrogen bonds and other noncovalent interactions that stably hold the two complementary DNA strands together. They use energy derived from the hydrolysis of nucleotide triphosphates for both bond breakage between complementary bases and translocation of a helicase enzyme along DNA. DNA unwinding activity catalyzed by a helicase usually exhibits a specific directionality (5′ to 3′ or 3′ to 5′) with respect to the DNA strand to which the enzyme is bound and moves. Unwinding activity of a DNA helicase and its related properties can be easily measured in vitro using common lab equipment. We will describe the detailed methods and notes for preparation of various helicase substrates and in vitro helicase assays using the substrates prepared.