Plant microRNA (miRNA) processing requires at least two cleavage steps of respective precursors. The first cleavage step is from pri-miRNA to pre-miRNA, and the second cleavage step is from pre-miRNA to mature miRNA. Using northern blot analysis, we previously showed that the RNase III enzyme Dicer-like protein 1 (DCL1) and the double-stranded RNA-binding (DRB) protein HYL1 are involved in the processing reactions of miRNA precursors. The processing of plant miRNAs differs from that of animal miRNAs in some respects. Here, we introduce our methods for analyzing the processing of miRNA precursors through transient expression of mutated pri-miRNAs with modified stem-loop structures in Nicotiana benthamiana. The methods described here could be useful for understanding how DCL proteins and cognate DRB proteins are involved in adequate recognition and processing of substrate precursor RNA molecules.