Microplastics (MPs) have become widespread pollutants in freshwater ecosystems, posing a considerable threat to aquatic and terrestrial life, including humans. The absence of standardized extraction and identification methods for MPs is impeding research progress. Current protocols for MP analysis face technical limitations in the context of analysis time, recovery rate, low detection limits, and errors due to manual sample handling, especially when dealing with diminutive-sized (<0.5 mm) particles. There is a necessity for constant improvement in current extraction and identification techniques, leading to the development of new methods for analyzing MPs. This chapter aims to provide an extensive overview of the most recent separation and analytical techniques for MPs, emphasizing their benefits and limitations. We have elaborately described the principles and applications of seven density-independent separation methods, three advanced microscopic imaging techniques, and four spectrometry-based elemental analyses for MP research in freshwater ecosystems. Separation of MPs with the assistance of magnets, electrostatic energy, oils, organic solvents, coagulants, and frothing agents were described. Characterization using high-definition microscopes, laser-induced spectroscopy, nuclear magnetic resonance, thermal cracking, and inductively coupled mass spectroscopy were addressed. Additionally, we presented algorithmic model-based hybrid approaches, which can be prioritized for future studies to potentially eliminate human error when dealing with minuscule particles. Implementation of the new methods can open up new avenues of MP analysis by providing precise and high-quality research output, although they have been overlooked by the scientific communities. This chapter encourages researchers to select efficient separation and identification methods for MPs research on freshwater ecosystems. Despite advancements, challenges persist in this domain, and this chapter facilitates the necessity for further development of innovative methodologies to enhance the detection and characterization limits, followed by accurate and reliable MP analysis.