After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using lambda exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.