Sulfamethoxazole compound, a combination of sulfamethoxazole and trimethoprim, is commonly prescribed to organ transplant recipients for infections caused by Nocardia, Burkholderia cepacia, and Pneumocystis jirovecii. The intricate pharmacokinetics and pharmacodynamics of this compound pose challenges in achieving optimal therapeutic drug levels. Therefore, monitoring the plasma concentration of this compound drug is necessary. To address this need, we have developed a rapid and straightforward dual-quaternary two-dimensional high-performance liquid chromatography (2D-HPLC) method for the simultaneous quantification of sulfamethoxazole and trimethoprim. This technique involves a one-step protein precipitation procedure, with the initial stage utilizing a solid-phase extraction column followed by an analytical column for the subsequent stage. Detection is achieved using a dual-quaternary 2D-HPLC system. Calibration curves for both sulfamethoxazole and trimethoprim were constructed, and the method underwent thorough validation in accordance with the Chinese Pharmacopoeia (Ch.P) standards, demonstrating remarkable accuracy and precision. This technique has been effectively employed for routine therapeutic drug monitoring (TDM) in organ transplant patients and other individuals requiring medical intervention.