The detrimental impact of microplastics (MPs) in natural water on human health and the environment has directed considerable attention toward the development of the corresponding quantification methods. However, the accurate quantification of MPs in natural water requires a meticulous sample pretreatment to distinguish them from other particulates. To address this need, we developed a rapid and simple pretreatment procedure based on heat- and base-activated persulfate oxidation, coupled with fluorescence microscopy. The pretreatment conditions were optimized by adjusting the reaction temperature and base concentration. The brief (15 min) persulfate treatment effectively degraded nonplastic microparticles (non-MPs) but minimally affected the physicochemical properties of polyethylene (PE)-MPs. Consequently, pretreating a mixture of PE-MPs and non-MPs in deionized (DI) water and various natural waters enabled the selective quantification of PE-MPs. Furthermore, the physicochemical properties of polypropylene (PP), polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA)-MPs, and UV-aged PE-MPs underwent negligible changes during the persulfate treatment, thereby indicating the feasibility of using the method across various types of MPs. In addition, the properties of MPs in natural water were simulated by considering the formation of biofilms and the adsorption of impurities, both of which can interfere with MP analysis. The activated persulfate oxidation separated bioaggregated MPs into individual particles by degrading the biofilms and removed adsorbed impurities, such as natural organic matter and clay minerals, from the surfaces of MPs.