Minicells, which are anucleate cells generated by irregular cell division, are emerging as promising drug delivery systems owing to advances in synthetic biology. However, their development is largely limited to a few model bacteria, highlighting the need to explore minicell platforms in alternative hosts. Lactiplantibacillus plantarum (L. plantarum), a probiotic bacterium classified as Generally Recognized as Safe, is an ideal candidate for such exploration. Minicell-producing L. plantarum was engineered by deleting the putative minD gene via plasmid-mediated homologous recombination, which inactivates cell division to form spherical minicells. Anucleate cells were isolated through differential centrifugation and filtration, followed by additional drug treatment to completely eliminate progenitor cells. Microscopy and flow cytometry analyses of the purified sample confirmed the absence of progenitor cells by DAPI staining. This protocol effectively produces bacterial minicells from L. plantarum for use in various biotechnological applications, including therapeutic agent delivery.