A plethora of biological barriers, intended to defend tissues and cells against external influences, stand in the way of efficient nucleic acid delivery by non-viral nanoplexes. Even when nanoplexes successfully evade extracellular barriers and reach their target cell, many intracellular barriers remain to be conquered. These include overcoming the plasma membrane, evading endosomal compartmentalization, and in some cases crossing the nuclear envelope. At the same time, exocytosis, autophagy and cytoplasmic degradation of the cargo should be avoided. Currently, there is a growing appreciation that the interaction of nanoplexes with these barriers should be understood in detail in order to rationally design a second generation of non-viral nanoplexes, capable of overcoming these many hurdles. Studying intracellular biobarriers is, however, quite challenging and specialized methods are constantly being developed. In this review, we present an overview of established as well as emerging techniques and assays that are currently available to the experimentalist to study nanoplex-barrier interaction, with a focus on quantitative methods. (C) 2018 Elsevier Ltd. All rights reserved.