The investigation of environmental nano- and submicron particles is needed for the assessment of their impact on the environment and human health as well as for understanding various natural and anthropogenic processes. Nano- and submicron particles have an increased mobility, may serve as a “carrier” for toxic and nutrient substances, and hence are of particular interest. So far, there is a lack of knowledge about source, behavior, fate, and toxicity of environmental nano- and submicron particles. This article is focused on the separation and characterization methods, which are currently used for their investigation. The application of sedimentation, centrifugation, membrane filtration, and field- and flow-based techniques to the separation of nano- and submicron particles are discussed. The advantages and limitations of the techniques are briefly summarized. Among characterization/analysis methods, a special attention is given to electron microscopy, light scattering as well as atomic absorption spectroscopy, optical emission, and mass spectrometry.