Delhi Institute of Pharmaceutical Sciences and Research, DPSRU, New Delhi-110 017, India Institute of Nuclear Medicine and Allied Sciences (INMAS), DRDO, Timarpur, New Delhi-110 054, India Chitkara University, Rajpura-140 401, Punjab, India E-mail: nidhisandal@rediffmail.com Manuscript received online 25 June 2018, revised 19 March 2019, accepted 20 March 2019 Insoluble Prussian Blue (PB) is a well established antidote for removal of cesium and thallium and its radioisotopes. Radiogardase-Cs is the only commercially available, capsules formulation of PB. Although PB is an intense blue colored powder but being an insoluble drug, quantitative estimation of PB is a challenge for researchers. Therefore, present research article is focused on the development of a new, simple and sensitive spectrophotometric method for determination of PB in bulk drug samples as well as formulations like Radiogardase. The method is based on two-step dissolution process. PB reacts with dilute base to form iron(III) hydroxide which reforms ferric hexacyanoferrate on reacting with acid. The resulting solution forms nanosuspension of PB which do not aggregate, and remain suspended in the aqueous solution and shows an absorption maxima at 710 nm. The reformed PB was characterized by infrared spectroscopy and particle size distribution analysis. Based on the UV-Visible spectral studies, analytical method for the determination of PB was developed and requisite validation parameters were performed. A linear response was observed in the range of 0.1–100 µg/ml with a regression coefficient of 0.9997. The LOD and LOQ were found to be 0.099 and 0.330 µg/ml, respectively.