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 (M-w), 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 an 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.