Lignocelluloses are widely investigated as renewable substrates to produce biofuels, e.g., ethanol, methane, hydrogen, and butanol, as well as chemicals such as citric acid, lactic acid, and xanthan gum. However, lignocelluloses have a recalcitrance structure to resist microbial and enzymatic attacks; therefore, many physical, thermal, chemical, and biological pretreatment methods have been developed to open up their structure. The efficiency of these pretreatments was studied using a variety of analytical methods that address their image, composition, crystallinity, degree of polymerization, enzyme adsorption/desorption, and accessibility. This paper presents a critical review of the first three categories of these methods as well as their constraints in various applications. The advantages, drawbacks, approaches, practical details, and some points that should be considered in the experimental methods to reach reliable and promising conclusions are also discussed.