The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarkers. However, one of prerequisites for the application of miRNAs in clinical practice is to accurately profile their expression. Currently available assays normally require pre-enrichment, amplification, and labeling steps, and most of them are semiquantitative. In this study, we converted the signal of target miR-21 into reporter peptide by a DNA-peptide probe and the reporter peptide was ultimately quantified using LC-MS/MS-based targeted proteomics. Specifically, substrate peptide GDKAVLGVDPFR containing reporter peptide AVLGVDPFR and tryptic cleavage site (lysine at position 3) was first designed, followed by the conjugation with DNA sequence that was complementary to miR-21. The newly formed DNA-peptide probe was then hybridized with miR-21, which was biotinylated and attached to streptavidin agarose in advance. After trypsin digestion, the reporter peptide was released and monitored by a targeted proteomics assay. The obtained limit of quantification (LOQ) was 1 pM, and the detection dynamic range spanned ∼5 orders of magnitude. Using this assay, the developed quasi-targeted proteomics approach was applied to determine miR-21 level in breast cells and tissue samples. Finally, qRT-PCR was also performed for a comparison. This report grafted the strategy of targeted proteomics into miRNA quantification.