Water systems are closely coupled with the terrestrial environment; they provide a key pathway for the infiltration of pollutants. Microplastics (MPs) are widely acknowledged as a dangerous threat to all forms of life. Overuse, improper release, and mismanagement of microplastics lead to their ingestion, accumulation, and transfer through different trophic levels and gradually pose a health risk to all the organisms at different levels. Various methods have been standardized for microplastic sampling, extraction, separation, visualization, identification, and characterization. The most widely accepted method of microplastics processing is the digestion of the samples using hydrogen peroxide as an oxidizing agent, characterization by Fourier Transformed Infrared Spectroscopy (FTIR), scanning electron microscope (SEM), fluorescence microscopy, Raman spectrometry, and Pyr-GC-MS. Most of the methods and techniques have shown promising results. Microplastics persist and accumulate in the environment for a long period of time as they undergo a very slow degradation process. The degradation process may depend on multiple variables existing in the surrounding environment and the composition of different types of polymers. This chapter provides a comprehensive discussion about sample collection using specific equipment, chemical extraction of MPs, the Nile red staining method, and light and dark contrast microscopy to isolate, quantify, and characterize the MPs from water, soil, and tissue samples. It also highlights the challenges that have been faced throughout the MP’s research spectrum and offers brief recommendations and suitable countermeasures to improve our understanding of MP extraction and isolation.