The present RP-HPLC method for in vivo estimation of furosemide in male rabbits was developed to improve its efficiency. The method was developed as per ICH guidelines. ODS column (C18 250 x 4.6 mm, 5µm) with flow rate 1 mL/min was used and the column temperature was set at 250C. The runtime was set for 10 min and the detection was done at λmax 210 nm. The mobile phase used was phosphate buffer (pH 4.6) and acetonitrile in 30:70 ratio. The developed method had shown high specificity for furosemide in rabbit plasma and the regression was found to be R2=0.9999 in the range of 0.1 to 5µg/0.5mL. The regression equation was found to be Y=0.4818X+0.0039. The lower limit of detection was found to be 0.1µg/0.5mL of plasma. The developed method for furosemide in rabbit plasma had shown good accuracy without any intra-day and inter-day variation. The proposed method was found to be adequate and reproducible for the estimation of furosemide in rabbit plasma.