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Rational synthesis and simple methodology for the purification of large (35-45 nm in lateral size) and flat (1.0-1.5 nm of height) nitrogen-doped graphene oxide quantum dots (GOQDs) are presented. The methodology allows robust metal-free and acid-free preparation of N-GOQDs with a yield of about 100% and includes hydrothermal treatment of graphene oxide with hydrogen peroxide and ammonia. It was demonstrated that macroscopic impurities can be separated from N-GOQD suspension by their coagulation with 0.9% NaCl solution. Redispersible in water and saline solutions, particles of N-GOQDs were characterized using tip-enhanced Raman spectroscopy (TERS), photoluminescent, XPS, and UV-VIS spectroscopies. The size and morphology of N-GOQDs were studied by dynamic light scattering, AFM, SEM, and TEM. The procedure proposed allows nitrogen-doped GOQDs to be obtained, having 60-51% of carbon, 34-45% of oxygen, and up to 7.2% of nitrogen. The N-GOQD particles obtained in two hours of synthesis contain only pyrrolic defects of the graphene core. The fraction of pyridine moieties grows with the time of synthesis, while the fraction of quaternary nitrogen declines. Application of TERS allows demonstration that the N-GOQDs consist of a graphene core with an average crystallite size of 9 nm and an average distance between nearest defects smaller than 3 nm. The cytotoxicity tests reveal high viability of the monkey epithelial kidney cells Vero in the presence of N-GOQDs in a concentration below 60 mg L -1 . The N-GOQDs demonstrate green luminescence with an emission maximum at 505 nm and sedimentation stability in the cell culture medium., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)