Introduction: Irregularities and defects on NiTi endodontic instruments originating from the manufacturing process can lead to the structural collapse and fracture of these instruments during treatment. To assess the cause of instrument wear and fracture, as well as increasing fracture incidence, destructive and non-destructive methods have been used for the analysis of surfaces and internal structures of new and used NiTi instruments. The aim of this systematic review was to undertake a detailed analysis of the methods used to evaluate the surface and internal microstructure of endodontic instruments. Methods and Materials: The scientific literature was comprehensively and systematically searched in the MEDLINE (PubMed), Web of Science, Cochrane Library, Scopus, and LILACS/BBO databases for studies published up to June 9, 2019. The eligibility criteria was based on the PICO (Patient, Intervention, Comparison, and Outcome) strategy with the question “ What is the best method for structural analysis of endodontic files? ” Two aspects were considered for inclusion in this study: (i) endodontic instruments and (ii) methods for structural analysis of NiTi instruments. Results: Based on the inclusion criteria, 94 articles were selected. The results showed that although specific methods have been used for qualitative and/or quantitative structural analysis of NiTi instruments, no study addressed both the surface and internal structure of the instruments at the same time. According to this review, the need to compare the methodologies used in the selected articles has been identified; however, each type of method used has its own limitation on the analysis of both the surface and the internal structure of the instruments. Conclusions: The comparison between the different types of methodologies used in the studies revealed the reliability and the limitations of the methods employed for structural analysis of endodontic instruments; thus assisting us in determining their validity.