Freshwater systems provide key pathways for microplastic (MP) pollution, and although existing studies have demonstrated the susceptibility of freshwater biota to ingestion, translocation, and trophic transfer, specific challenges pertaining to methodological standardization remain largely unresolved, particularly with respect to isolating, characterizing, and assessing MPs. Here, a critical review is performed outlining the challenges and limitations currently faced by freshwater MP researchers, which may well apply across the MP research spectrum. Recommendations are provided for methodological standardization, particularly in MP characterization, quality assurance, and quality control (QA/QC) procedures as well as reporting. Considerations for the assessment of MPs in freshwater biota as a means of improving comparisons between studies are discussed. Technological advancements, including the improvement of laboratory infrastructure for identifying MPs within the smaller size range as well as methodological standardization are essential in providing policy makers with tools and measures necessary to determine the distribution of MPs within freshwater ecosystems, while also allowing for comparability and providing compliance for future monitoring requirements.