Replication fork helicases unwind DNA at a replication fork, providing polymerases with single-stranded DNA templates for replication. In bacteria, DnaB unwinds DNA at a replication fork, while in archaeal and eukaryotic organisms the Mcm proteins catalyze replication fork unwinding. Unwinding in archaea is catalyzed by a single Mcm protein that forms multimeric rings, whereas eukaryotic helicase activity is catalyzed by the heterohexameric Mcm2-7 complex acting in concert with Cdc45 and the GINS complex. A subcomplex of eukaryotic Mcm proteins, the Mcm4,6,7 complex, unwinds DNA in vitro, and studies of this assembly reveal insight into the mechanism of the eukaryotic Mcm helicase. Detailed methods for the investigation of replication fork helicase mechanism are described in this chapter. Described herein are methods for the design of DNA substrates for unwinding and branch migration studies, annealing DNA, purifying replication fork helicase proteins, and analyzing DNA unwinding activity.