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The most frequently used method to quantify microplastics (MPs) visually by microscope is time consuming and labour intensive, where the method is also hindered by the size limitation at 10 µm or even higher. A method is proposed to perform pre-concentration of MPs by vacuum filtration, hydrogen peroxide wet digestion, fluorescent staining and flow cytometric determination to rapidly detect and quantify small MPs sized from 1–50 µm. The method performance was evaluated by the spiking of seven different types of polymer, including polystyrene (PS), low-density polyethylene (LDPE), polypropylene (PP), poly(methyl methacrylate) (PMMA), polyvinyl chloride (PVC), polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS) at different levels (400, 4000, 40,000 particles mL−1), with a satisfactory overall % recoveries (101 ± 19.4%) observed, where in general no significant difference between the two methods was observed. Furthermore, a pre-concentration process by vacuum filtration was introduced to reduce the matrix effect. After pre-concentration, satisfactory % recoveries and accuracy in MP counts resulted from both ultrapure water (94.33 ± 11.16%) and sea water (103.17 ± 9.50%) samples. The validated method using flow cytometry can be used to quantify MPs in environmental water samples that can reduce time and human resources.