The analyses of thermochemically-derived bio-oil properties and composition are challenging due to the diversity of compounds present and the reactivity of the oils. There are currently a variety of techniques used and no standard method established for the analysis of the molecular weight distribution, weight average molecular weight (Mw) and other molecular weight metrics of bio-oils. This review focuses on the challenges and variation in methodologies employed for the analysis of bio-oils on the basis of molecular weight, particularly by gel permeation chromatography (GPC). GPC is the most practical means for determination of molecular weight metrics of bio-oils but needs to be refined using appropriate standards and/or detectors to ensure consistency and accurate quantification of molecular weight metrics. Future method development for a robust technique with accurate and comparable molecular weight data should focus on GPC with multiple detection methodology on whole bio-oils, verified relative to another technique such as mass spectrometry (MS). MS techniques, such as Fourier transform-ion cyclotron resonance (FT-ICR MS), have also been utilized for the determination of molecular weight distribution of bio-oils and are briefly addressed in this review. Many MS methods can provide extensive characterization and structural speciation of components in bio-oils, and while accurate molecular weight metrics can be obtained with the appropriate use of ionization techniques and optimized parameters to ensure appropriate range of m/z and signals representative of abundance, MS is not a robust or an economically practical method for routine molecular weight analyses. Physical separation techniques such as preparative scale GPC, distillation, and liquid-liquid extraction methods are also briefly addressed in this review in the context of molecular weight analyses.