Objective To evaluate the acute oral toxicity of ethyl acetate fraction of Sesbania grandiflora Linn. roots as per OECD 425 guidelines. Methods The two groups of five female albino mice were selected and both the groups of mice were observed for 14 days for various parameters according to OECD guidelines 425 guidelines after treating one group as a normal control and other with ethyl acetate fraction of Sesbania grandiflora root EFSGR 2000mgkg p.o.Results Oral dose of EFSGR 2000mgkg did not resulted in death and treatment related adverse effects such as modification in food and water consumption body weight variation relative organ weights biochemical and hematological parameters. But treatment showed mild changes in behavioral patterns like sleeping and itching during initial time point. Further histopathological evaluation of vital organs exhibited normal architecture and this observation provided good support to general and biochemical measurements.Conclusion The current study showed that the ethyl acetate fraction of Sesbania grandiflora did not exhibit any signs of toxicity in female albino mice. Hence proved that the toxicity study based calculated dose of EFSGR can be used for the pharmacological studies.