The discovery of microRNAs (miRNAs) has opened an entire new avenue for drug 
development. These short (15-22 nucleotides) noncoding RNAs, which function in 
RNA silencing and posttranscriptional regulation of gene expression, have been 
shown to critically affect numerous pathways in both development and disease 
progression. Current miRNA drug development focuses on either reintroducing the 
miRNA into cells through the use of a miRNA mimic or inhibiting its function via 
use of a synthetic antagomir. Although these methods have shown some success as 
therapeutics, they face challenges particularly with regard to cellular uptake 
and for use as systemic reagents. We recently presented a novel mechanism of 
inhibiting miR-544 by directed inhibition of miRNA biogenesis. We found that 
inhibition of DICER processing of miR-544 through the use of a small molecule 
abolished miR-544 function in regulating adaptation of breast cancer cells to 
hypoxic stress. Herein, we describe a protocol that utilizes bioinformatics to 
first identify lead small molecules that bind to DICER cleavage sites in 
pre-miRNAs and then employ an efficient, high-throughput fluorescent-based 
screening system to determine the inhibitory potential of the lead compounds and 
their derivatives.