Abstract Single-molecule characterization of protein-DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information for fluorescently tagged proteins isolated from nuclear extracts of human cells. This approach determines binding lifetimes ( k off ), events per second (∼ k on ), positional dependence (specificity), and characterizes 1D diffusion along DNA. We demonstrated the wide applicability of this approach on three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), xeroderma pigmentosum complementation group C protein (XPC), and 8-oxoguanine glycosylase 1 (OGG1). By measuring multiple fluorescent colors simultaneously, we additionally characterized the assembly and disassembly kinetics of multi-protein complexes on DNA. Thus, Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE) provides new insights about damage recognition and represents a universal technique that can be used to rapidly characterize numerous protein-DNA interactions.