Microplastic contamination of the marine environment has been reported globally. Its pervasiveness has highlighted the importance of accurate quantification to enable comparability within and between different environmental matrices. The potential efficacy of different methods to separate microplastics from their environmental sample matrix, however, is rarely validated. In this study, we examine the effects of four commonly used separation methods for seawater samples, namely, visual separation, density flotation, acidic digestion, and enzymatic digestion, using high-density polyethylene as our model microplastic. For each separation method, clarification efficiencies of the sample matrix, spiked recovery of high-density polyethylene microparticles, and potential changes in the chemical and physical characteristics of high-density polyethylene were assessed. High, albeit variable, recovery rates (>83%) of high-density polyethylene microparticles were achieved across all methods. Concentrated nitric acid was most effective at eliminating biological material from seawater samples. No apparent physical (i.e., length or color) or chemical changes due to separation treatments were observed in recovered high-density polyethylene microparticles, with the one exception that enzymatic digestion obscured polymer identification of high-density polyethylene. Our findings highlight the need to determine and report on the accuracy of separation methods for different polymer types and specific environmental sample matrices to ensure accurate quantification of marine microplastic contamination.