Plants and animals microRNAs (miRNAs) have been proposed to be key regulators of many fundamental processes. However defining miRNAs function has been problematic due to the paucity of miRNA loss-of-function mutants. This is likely due to their small gene size and redundancy as most miRNA have highly related family members. Consequently, the analysis of miRNA function has been primarily based on predictive bioinformatic or transgenic gain-of-function approaches. However, a number of new methodologies have been developed able to result in loss-of-function phenotypes. This includes miRNA sponges in animals and target mimicry in plants, both of which sequesters the mature miRNAs, disrupting endogenous miRNA:mRNA target relationships. Furthermore, artificial miRNAs and RNA interference in plants have been shown to be potent silencers of MIRNA genes. We will discuss the strengths and weaknesses of these methodologies which are potentially of great biotechnological use in medicine and agriculture.