This experimental manual provides comprehensive information about the designing and execution of the experimental protocols for radioprotective drug screening. The manual divided into seven sections. Each section arrange in chronological order from preliminary to advance methods to evaluate a drug molecule for its probable radioprotective properties. This manual is first of its kind in the area of radiation biology and radioprotection. Preliminary in vitro assay (section I) designed to evaluate in vitro antioxidant activity of test compound which are integral part of radioprotection. Secondary methods (Section II) designed to evaluate radioprotective efficacy of test compound towards biomacromolecules i.e. proteins, enzymes and plasmid DNA. The third section (Section III) of this manual dedicated to evaluate in vitro radioprotective potential of test compound in cell culture in terms of colony forming unit (CFU), superoxide dismutase, catalase, glutathione reductase, glutathione-s-transferase, and lysosomal membrane stability. Radiation induced gemonic DNA damage and apoptosis is the common cause of cell death in cells. Therefore, to assess DNA damage and apoptosis inhibitory activity of the test compound a complete section (section IV) was formulated. Further, to estimate mitochondrial DNA damage and perturbation in oxidative phosphorylation as well its modulation by radioprotective drug molecule detailed protocols were mentioned in section V and section VI of the manual. Last but not the least, section VII of this manual was designed to evaluate immune system radioprotection by radioprotective compound. The manual arranged in systematic manner, by follow the protocols of this manual researcher can confirm and classify the radioprotective efficacy of the test molecule.