MicroRNAs (miRNAs) constitute a diverse class of small RNAs (sRNAs) exhibiting distinct expression patterns during pivotal biological processes in plants such as development and environmental responses. Precise quantification of miRNAs is imperative for unraveling their intricate functional roles. Although current real-time PCR techniques offer sensitivity and specificity, their widespread adoption is hindered by prohibitive costs, particularly for laboratories demanding high-throughput analyses with robust yet economically viable outcomes. Drawing inspiration from a methodology proven effective in miRNA quantification from human serum, this chapter outlines a novel real-time probe-based RT-PCR procedure tailored for plant miRNAs. The versatility of this method is showcased through successful application in plants, underscoring its cost-effectiveness and sensitivity. Our findings endorse this probe-based qPCR technique as a practical solution for affordable and accurate microRNA quantification in diverse plant species.