Plastics represent the largest component of marine debris globally. In this context, it is essential to quantify the current extent of plastic pollution, including microplastics (MP; plastics < 5 mm), within marine abiotic and biotic compartments. Despite significant effort, MP studies still face methodological impediments to establish accurate and standardized protocols to separate, process and analyze MPs in environmental samples. Furthermore, underestimation and overestimation of MP contamination, either through loss of MPs or introduction of extraneous MPs during handling and processing, is concerning, particularly when assessing risk profiles for marine ecosystems. Presented here is a custom-made stainless steel vacuum filtration apparatus designed to perform size-tiered separation and facilitate retrieval of MPs from a variety of environmental sample matrices. Incorporating this apparatus into a standard MP workflow achieved efficient graduated separation of commonly found MP fragments and fibers, validated by spike-recovery tests. As a case study, the gastrointestinal tracts of three juvenile Australian sharpnose sharks, Rhizoprionodon taylori, were processed using the filtration apparatus, and 46 anthropogenic items ranging from 0.021 to 8.87 mm were retrieved. This study demonstrates the effective use of the size-tiered stainless steel vacuum filtration apparatus and an improved efficiency in downstream microphotography and spectroscopic analyses of MPs from a complex sample matrix. Finally, it contributes to the MP research field by delivering more reliable estimates of MP contamination in marine ecosystems.