MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that bind to target mRNAs and regulate their expression. Recent evidence has indicated the involvement of miRNAs in human malignancies. It has been suggested that aberrantly down-regulated or up-regulated miRNAs may be replaced with synthetic miRNAs or antagomiRNAs, respectively, and restore normal cell functions. As therapeutic development requires analytical support, we developed and validated an ultrasensitive and selective assay for quantification of synthetic 2′-methoxyphosphorothiolate-miRNA in mouse plasma and cell lysate for the first time. The method is based on a hybridization-ligation fluorescence enzyme-linked immunosorbent assay and has provided a linear dynamic range of 10-1,000,000 pM for three synthetic miRNAs both singly and in a mixture. The intra- and inter-day coefficients of variation were <20% and the accuracy values nearly 100%. Using this assay, we performed pharmacokinetic studies of three synthetic miRNAs in mice treated with a single i.v. bolus dose of 7.5 mg kg-1. The 2-methoxyphosphorothiolate-miRNAs reached peak concentrations in the μM and nM ranges in plasma and bone marrow, respectively, and remained measurable at 24 h. These concentrations are in a range that shows biological activities. We conclude that this method provides a general and valuable tool for the pharmacologic study and clinical development of synthetic miRNAs.