Effective separation and purification of nanoparticles are crucial for their use in biomedicine, catalysis, environmental sensing, and materials science. Herein, this review provides a summary of the most important separation methods adjusted to separate nanoparticles according to size, shape, surface features, and magnetic responsiveness. Traditional methods such as centrifugation are common due to their simplicity but tend to be inefficient when dealing with low-density or fragile nanoparticles. Magnetic separation methods, such as MACS columns and MagBed systems, provide non-aggressive, scale-up solutions for isolating superparamagnetic nanoparticles. Size exclusion chromatography (SEC) offers non-destructive size-based separation, and ultrafiltration provides high-yield, quick purification with pore-selective membranes. More recent systems such as membrane-based filters with nanocrystalline or π-conjugated structures, hydrogel electrophoresis, and capillary electrophoresis enable high-resolution sorting according to electrophoretic mobility and steric interaction. Microfluidic platforms, active and passive, facilitate continuous high-throughput separation on the microscale. Monolithic columns and high-gradient magnetic separation also provide additional purification capacity for advanced nanoparticle formulations. The incorporation of artificial intelligence (AI) has added data-driven optimization, real-time control, and predictive modelling, greatly improving the efficiency and sustainability of nanoparticle separation processes. Collectively, these new methodologies provide robust, accurate, and scalable methods for realization of uniform, application-ready nanoparticle populations.