Abstract: LC-MS/MS methods for the quantitative determination of mecobalamin and ceftriaxone in rat plasma were established, and utilized to assess the pharmacokinetic behaviors of the two drugs in rats and to determine whether there were pharmacokinetics-based drug-drug interactions between them. Methanol was used as a protein precipitant to process rat plasma samples. For mecobalamin, acetonitrile containing 0.1% formic acid was used as the organic phase, while an aqueous solution of 0.1% formic acid and 2 mmol/L ammonium acetate served as the aqueous phase. For ceftriaxone, the organic phase was acetonitrile with 0.1% formic acid, and the aqueous phase was a 1% formic acid aqueous solution. LC-MS/MS quantitative analysis methods for both drugs were developed under the positive ion mode, followed by comprehensive methodological validation. Single-dose administration (either alone or in combination) was carried out via caudal vein injection. The dosing regimens were 0.03, 0.1, and 0.3 mg/kg for mecobalamin and 90 mg/kg for ceftriaxone. The results of methodological validation indicated that mecobalamin exhibited good linearity in the range of 3−3000 ng/mL (r = 0.9991), and ceftriaxone showed excellent linearity in the range of 0.5~500 μg/mL (r = 1). The selectivity, precision, accuracy, extraction recovery, matrix effect, and stability of the analytical methods for both substances met the relevant requirements. The pharmacokinetic study revealed that, compared with single-drug administration, the average plasma concentration-time profiles of the two drugs almost overlapped during single-dose combined administration. Statistical analysis, specifically one-way analysis of variance, demonstrated no significant differences in major pharmacokinetic parameters (t_1/2, AUC_0−∞, etc.) (P > 0.05). This study confirmed that no pharmacokinetic-based drug-drug interactions occurred when mecobalamin and ceftriaxone were co-administered in rats. The findings of this research offer a reliable pharmacokinetic foundation for the rational clinical co-use of these two drugs, thus providing valuable insights into optimizing therapeutic strategies.