Numerous methods have been developed for microplastics isolation and quantification in various environmental media, many of which require elaborate or expensive analytical equipment and decontaminated lab space. This study seeks to create a reproducible and economical method for isolating microplastics in surface water that rely on Nile Red staining. We use a Nile Red pre-staining step prior to sample digestion, density separation, and filtration to mitigate downstream in-lab contamination before quantification of microplastics via fluorescent microscopy. To test the method, we collected replicate surface water samples from several reaches of an urban stream in the Chesapeake Bay Watershed, USA seasonally for one year. The proposed sampling and quantification method found some success in these surface water samples with specific microplastics (≥20 μm) able to be enumerated.