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Fahad Albalawi,1,2,* Mohd Zobir Hussein,2,* Sharida Fakurazi,3,4 Mas Jaffri Masarudin5 1Department of Medical Laboratory and Blood Bank, King Fahad Specialist Hospital-Tabuk, Tabuk, Saudi Arabia; 2Materials Synthesis and Characterization Laboratory, Institute of Advanced Technology (ITMA), Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 3Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 4Natural Medicine and Product Research Laboratory Institute of Bioscience, Serdang, Selangor, Malaysia; 5Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia*These authors contributed equally to this workCorrespondence: Mohd Zobir Hussein Email mzobir@upm.edu.myAbstract: The emergence of nanotechnology as a key enabling technology over the past years has opened avenues for new and innovative applications in nanomedicine. From the business aspect, the nanomedicine market was estimated to worth USD 293.1 billion by 2022 with a perception of market growth to USD 350.8 billion in 2025. Despite these opportunities, the underlying challenges for the future of engineered nanomaterials (ENMs) in nanomedicine research became a significant obstacle in bringing ENMs into clinical stages. These challenges include the capability to design bias-free methods in evaluating ENMs’ toxicity due to the lack of suitable detection and inconsistent characterization techniques. Therefore, in this literature review, the state-of-the-art of engineered nanomaterials in nanomedicine, their toxicology issues, the working framework in developing a toxicology benchmark and technical characterization techniques in determining the toxicity of ENMs from the reported literature are explored.Keywords: engineered nanomaterials, nanomedicine, nanotoxicology, particle tracking analysis, asymmetric flow field-flow fractionation, Taylor dispersion analysis