Delivery of the drug to a desired point of body and controlled release of the therapeutic agent are important features, provided by drug delivery systems (DDSs), for development of today's effective medicines. A variety of nanomaterials or nanomolecules such as lipids/liposomes, nucleic acids, peptides/proteins, composites, polymers, or carbon nanotubes can be used as DDSs. Single-molecule characterization of these small materials in terms of their size, shape, surface, encapsulation efficiency, as well as interaction with the drug-receiving cell has importance for their efficiency. The loading, distribution, or leakage of the drug as well as its interaction with DDS should also be characterized. Although diverse techniques are present for characterization of specific DDS material, methods such as electron microscopy and fluorescence microscopy are widely used. In this review, the current methodologies utilized for the single-molecule characterization of mostly preferred DDS materials were presented.