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The objective of this study is to investigate the biodistribution of Indocyanine green (ICG) in healthy mice, when delivered through polymeric nanoparticles. The poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles entrapping ICG were engineered and characterized. The extraction method for ICG recovery from biological samples was developed. The biodistribution of ICG was determined in healthy C57BL/6 mice (female, 10-week old) when delivered through PLGA nanoparticles in comparison to free ICG solution, using a fluorometric assay method. The extraction method for ICG shows efficiency above 80% for various organs and plasma. When nanoparticles were used to deliver ICG, 2–8 times higher concentrations of ICG was deposited in various organs, with 5–10 times higher plasma levels till 4 h, after an i.v. dose as compared to free ICG solution. In conclusion, the nanoparticle formulation significantly increased the ICG concentration and circulation time in plasma as well as the ICG uptake, accumulation and retention in various organs. Overall, this study represents the first step in exploring and establishing the potential of nanoparticles as an ICG-delivery system for use in tumor-diagnosis and photodynamic therapy.