With increased application of Next Generation Sequencing (NGS) in the diagnosis of monogenic Ehlers-Danlos syndromes, there is an increased probability to identify variants of unknown significance. Additionally, in some cases no genetic alteration may be identified whilst there is a strong clinical suspicion on a monogenic EDS type. The diagnostic value of non-genetic investigations, which prior to NGS were quite commonly used to support the clinical diagnosis of monogenic EDS types, is explored. In addition, new structural/functional investigations that could deliver evidence towards pathogenicity are discussed. It appears that certain functional and/or structural investigations used frequently in the past can remain helpful and can provide additional evidence that may confirm a clinical diagnosis of a monogenic EDS type. However, there is a need for the development of novel structural/functional studies for monogenic types of EDS. The level of evidence of such studies for application in the established diagnostic DNA variant classification criteria remains to be determined.