A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completelyin vitroapproach for the characterization of DNA regulatory elements usingEscherichia coliextract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performancein vivofor the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completelyin vitromethod to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. Thisin vitroapproach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology.