Classical biochemical approaches have made great contribution to our current understanding of how small interfering RNA (siRNA) and microRNA (miRNA) are produced and function. However, it has been challenging to establish in vitro systems that can dissect the mechanism of PIWI-interacting RNAs (piRNAs), largely due to the lack of suitable cultured cell lines possessing the piRNA pathway endogenously. The silkworm ovary-derived BmN4 cell line is an emerging model for studying piRNAs, because this cell line harbors a fully functional germline piRNA pathway. We recently reported in vitro recapitulation of a part of piRNA biogenesis-loading of precursor piRNAs and 3'-end maturation-by using lysate prepared from BmN4 cells. Here we describe the methods for maintenance of BmN4 cells, preparation of cell lysate, and in vitro assays to dissect piRNA biogenesis.