The cytoplasmic membrane is one of the most frequent cell targets of antimicrobial peptides (AMPs) and other biomolecules. Understanding the mechanism of action of AMPs at the molecular level is of utmost importance for designing of new membrane-specific molecules. In particular, the formation of pores, the structure and size of these pores are of great interest and require nanoscale resolution approaches, therefore, biophysical strategies are essential to achieve an understanding of these processes at this scale. In the case of membrane active peptides, pore formation or general membrane disruption is usually the last step before cell death, and so, pore size is generally directly associated to pore structure and stability and loss of cellular homeostasis, implicated in overall peptide activity. Up to date, there has not been a critical review discussing the methods that can be used specifically for estimating the pore dimensions induced by membrane active peptides. In this review we discuss the scope, relevance and popularity of the different biophysical techniques such as liposome leakage experiments, advanced microscopy, neutron or X-ray scattering, electrophysiological techniques and molecular dynamics studies, all of them useful for determining pore structure and dimension. Copyright © 2021. Published by Elsevier B.V.