R-loops, three-stranded RNA–DNA hybrids that arise mostly during transcription, could cause genomic instability via distinct routes. Detection of genomic RNA–DNA hybrids via immunofluorescence and RNA–DNA hybrid immunoprecipitation techniques have facilitated the discovery of many cellular factors that maintain R-loop homeostasis. One of multiple R-loop avoidance mechanisms is mediated by several nucleic acid motor proteins that utilize the energy from ATP hydrolysis to dissociate the R-loop structure. The biochemical activity of such motor proteins can be interrogated using synthetic R-loop substrates. Here, we describe methods to generate R-loop and RNA–DNA substrates for studying the activity of R-loop processing motor proteins such as human DHX9 and S. cerevisiae Pif1. Such studies provide valuable information regarding the directionality, nucleic acid strand preference, and processivity of these enzymes. Moreover, these substrates and companion biochemical assays provide the requisite tool for understanding the action of physiologically relevant regulators of these motor proteins.