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The study was aimed to validate and optimize high performance liquid chromatographic (HPLC) method for the determination of coumarin-3-carboxylic acid (C3A) in the heart and liver issue of Sprague-Dawley (SD) rats after intragastric administration of extractive of leaves of Ficus virens var sublanceolata. And simple ADME and target prediction analyses were performed for C3A. Ethyl acetate was employed to precipitate protein with appropriate sensitivity and acceptable matrix effects. The satisfactory separation was developed on an ODS2 column (4.6 mm × 250 mm, 5 μm) by gradient elution with a methanol-acetic acid solution (pH = 3.0) as the mobile phase. The flow rate was 1.0 mL min−1, the column temperature was maintained at 30 ± 2 °C, the injection volume was 20 μL, and the detection wavelength was set as 309 nm. The method was fully validated in terms of selectivity, linearity, accuracy, precision, extraction recovery and stability. The results of the ADME analysis found that C3A has excellent characteristics of drug-likeness, consistent with good bio-absorption. And the predicted 12 target protein belongs to the amine oxidoreductase and carbonic anhydrase target class. This method is simple, rapid, sensitive, and accurate for the determination of coumarin-3-carboxylic acid in the heart and liver tissue of SD rats.