A high-performance liquid chromatography method coupled with diode array detection (HPLC-DAD) was developed for the simultaneous quantification of andrographolide (AG) and 14-deoxy-11,12-didehydroandrographolide (DDAG) in rat serum. A salt-assisted liq-uid-liquid extraction (SALLE) procedure was optimized, with MgSO4 yielding the highest extraction efficiency (>90% for both AG and DDAG), outperforming conventional solvent extraction and comparable to solid-phase extraction. The method exhibited acceptable linearity (125–2000 ng/mL, r2 > 0.99), with low limits of detection and quantification of 60 and 70 ng/mL for AG and 201 and 234 ng/mL for DDAG, while adhering to the ICH M10 criteria for accuracy, precision, and stability under various storage conditions. Stability testing of the prepared samples demonstrated that > 99% of AG and 95% of DDAG were retained when stored at low temperatures, specifically below 4 °C. The developed method was successfully applied for a pharmacokinetic study following oral administration of Andrographis paniculata extract (containing AG 7.5 mg/kg) in healthy Wistar rats. The SALLE-HPLC-DAD developed herein enables selective AG quantification without signifi-cant matrix interference. In conclusion, this study introduces an alternate sample prepa-ration and analytical method that is fast, cost-effective, and reliable, making it suitable for pharmacokinetic studies of the principal biomarker of Andrographis paniculata.