Abstract: The accurate quantification of serum 25-hydroxyvitamin D (25(OH)D) is critical for the clinical assessment of vitamin D status. While liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold standard, imported instruments dominate the market. However, the high cost associated with these imported systems has significantly limited the broader application of LC-MS/MS in routine clinical settings. Domestically produced mass spectrometers, with their cost-effectiveness and localized service support, are now sufficiently competitive to challenge market dominance. This study aimed to comprehensively evaluate the analytical performance of a domestic LC-MS/MS system, the BGI LMSQ-1000, for the quantification of 25(OH)D₂ and 25(OH)D₃ in serum and to rigorously assess the agreement of the obtained results with a widely used imported system, the AB 4500MD. A full validation of the BGI LMSQ-1000 method was performed in accordance with the US FDA bioanalytical method validation guidance. Key parameters assessed included accuracy (expressed as % bias), precision (intra-assay and inter-assay coefficients of variation, CVs), limits of detection (LODs), and linearity. Subsequently, 145 serum samples were analyzed in parallel using both the BGI LMSQ-1000 and the AB 4500MD systems. The agreement of quantitative results between the two platforms was analyzed using Bland-Altman plots. Furthermore, clinical classification agreement (deficiency: <12 μg/L; insufficiency: 12-20 μg/L; sufficiency: ≥20 μg/L for total 25(OH)D) was evaluated using the weighted Kappa statistic. The BGI LMSQ-1000 system demonstrates satisfactory analytical performance. Accuracy is within ±20% for all QC levels for both 25(OH)D₂ and 25(OH)D₃. Precision is excellent, with intra-assay and inter-assay CVs of ≤5.21% and ≤3.78%, respectively. The LODs are determined to be 0.39 μg/L for 25(OH)D₂ and 0.74 μg/L for 25(OH)D₃, respectively. Linear calibration curves show good linearity in the ranges of 1.56-100.00 μg/L for 25(OH)D₂ and 2.95-189.00 μg/L for 25(OH)D₃, respectively. Method comparison reveals excellent agreement between the two instruments. Bland-Altman analysis showed that more than 95% of the differences between the BGI LMSQ-1000 and the AB 4500MD fall within the acceptable limits of agreement for both analytes. The weighted Kappa coefficient for clinical classification agreement reaches 0.95, indicating almost perfect agreement with the BGI LMSQ-1000 system. In conclusion, the domestic BGI LMSQ-1000 LC-MS/MS system exhibits reliable accuracy, precision, sensitivity, and linearity for the simultaneous quantification of serum 25(OH)D₂ and 25(OH)D₃. It shows outstanding methodological concordance and clinical classification agreement compared to the established AB 4500MD system. These findings validate that the BGI LMSQ-1000 is a competent analytical platform suitable for meeting the demands of clinical diagnostics and vitamin D status assessment.