Recent studies have shown that the microRNA (miRNA) pathway is an evolutionarily conserved endogenous pathway that is important for normal development. Mature miRNAs are excised from precursors in a stepwise process and subsequently incorporated into an RNA-induced silencing complex (RISC), which mediates either cleavage of the target messenger RNA (mRNA) or translational repression, depending on the complementarity between the miRNA and its target mRNA. In this chapter, we describe in vitro precursor (pre)-miRNA processing assays using Drosophila Schneider-2 (S2) cell lysates and immunopurified materials.