Analyses of gene functions in vitro provide the means to dissect biological phenomena. Development of cell-free assays for transcription, splicing, and translation has yielded great insights into macromolecular interactions and functions required for these processes. This article discusses development of cell-free assays to test macromolecular interactions and activities required for microRNA effector functions. This chapter also compares in vitro analyses to complementary studies in cells and the technical considerations that permit molecular analysis of microRNA function in cell-free conditions.