Abstract Plants are good sources of biologically active compounds with antimicrobial activity, including polypeptides. Antimicrobial peptides (AMPs) represent one of the main barriers of plant innate immunity to environmental stress factors and are attracting much research interest. There are some extraction methods for isolation of AMPs from plant organs based on the type of extractant and initial fractionation stages. But most methods are directed to obtain some specific structural types of AMPs and do not allow to understand the molecular diversity of AMP inside a whole plant. In this mini-review, we suggest an optimized scheme of AMP isolation from plants followed by obtaining a set of peptides belonging to various structural families. This approach can be performed for large-scale screening of plants to identify some novel or homologous AMPs for fundamental and applied studies.