This study aimed at the evaluation of plasma level profiles of choline produced after the oral administration of choline alphoscerate (CA). For this purpose, a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analytical method was developed and validated. Baseline correction technique was employed to monitor the CA-related choline in plasma by subtracting background levels of choline from total choline levels measured after the drug administration. Choline was well separated in the plasma matrix under the analytical conditions employed for HPLC/MS/MS and the analytical method developed for the monitoring of choline in plasma was well validated. The endogenous choline levels in plasma under fasting condition were measured to be 7.78 AE 1.74 μmol/L for test period 1 and 7.88 AE 1.78 μmol/L for test period 2, respectively, and were considerably increased after consumption of meals. The corrected choline levels in plasma produced only by the oral administration of CA could be evaluated by subtracting the background levels of choline monitored before the drug was administered. The pharmacokinetic parameters estimated for test and control formulations were quite similar based on the comparison of C max , AUC 12h , and T max , indicating that these are biologically equivalent dosage forms. The results of our study consequently suggest that the baseline correction method in evaluating plasma concentration profiles of endogenous substances is useful.