MicroRNAs are increasingly recognised as important down-regulators of gene expression. Luciferase-based assays have traditionally been used for identifying microRNA targets but these are limited. Previously we have reported miR-212 targeting of the Kir2.1 3′ UTR using a novel functional microRNA targeting assay and here we report targeting by miRs-15b, -132 and -424. These microRNAs have been shown to be upregulated in heart failure, which features downregulation of Kir2.1 activity in its pathophysiology. In the assay the 3′ UTR of the inward rectifier K+ channel Kir2.1 was inserted downstream of the mCherry red fluorescent protein sequence in an expression plasmid. MicroRNA, siRNA control or non-targeting control (SCR) sequences were inserted into the pSM30 expression vector, which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were transiently co-transfected with the pSM30- and mCherry-based plasmids and the mean red and green intensity of each cell was measured using Volocity software. The principle of the assay is that functional targeting of the 3′ UTR by the microRNA decreases the cell red/green fluorescence intensity ratio. It was validated with a siRNA targeting the 3′ UTR of Kir2.1 and used to investigate targeting of the Kir2.1 3′ UTR by miR-15b, miR-132 and miR-424 as predicted by bioinformatics. Red/green intensity ratio was significantly lower in siRNA-expressing and microRNA-expressing cells vs. SCR-expressing controls (p<0.001 for all microRNAs and siRNA by ANOVA; n range 322 to 2935). Similar results were obtained in 3 independent experiments for each microRNA. These results indicate functional targeting of the 3′ UTR of Kir2.1 by miR-15b, miR-132 and miR-424, and suggest their involvement in Kir2.1 downregulation in heart failure.