In plants, RNA interference pathways (RNAi) play a critical role in defense against viruses and other pathogens, as well as in the regulation of developmental processes. Research in the plant RNA silencing field over the last two decades has focused on identifying the main players and their mechanistic roles. However, understanding the spatiotemporal and subcellular distribution of RNA molecules has been persistently challenging limiting our understanding of RNA-guided processes in plants. In the past, we demonstrated that ARGONAUTE1 (AGO1), the main effector of the microRNA (miRNA) pathway, is capable of loading miRNA in the nucleus, and subsequently translocating miRNA from the nucleus to mediate the miRNA-directed cleavage of target mRNAs in the cytoplasm. Nevertheless, our recent findings strongly indicate that miRNAs are also able to diffuse from the nucleus to the cytoplasm independently of AGO1. Here, we present a novel approach for nuclear sorting via flow cytometry, incorporating essential checkpoints to ensure the high purity of isolated nuclei. Using this method, we investigated the subcellular distribution of small non-coding RNAs in Arabidopsis. Additionally, we examined the subcellular localization of miRNAs in both wild-type and hst-1 mutant plants to evaluate the potential involvement of HASTY in sRNA nucleocytoplasmic transport.