Dibenzoyl-L-tartaric acid (L-DBTA) is a crucial compound in the synthesis of chiral molecules, particularly within the pharmaceutical industry. Ensuring the enantiomeric purity of L-DBTA is essential for regulatory compliance, quality control, and process optimization. To achieve this, a high-performance liquid chromatography (HPLC) method was developed and validated for determining the D-DBTA content in L-DBTA. The method validation adhered to ICH Q2(R2) guidelines, covering parameters such as system suitability, solution stability, robustness, linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision. HPLC separation was performed using a Chiral PAK IA column (250 × 4.6 mm, 5.0 μm) with an isocratic mobile phase consisting of n-heptane, isopropanol (IPA), and trifluoroacetic acid (900:100:1 v/v/v). The column temperature was maintained at 40°C, and the sample cooler was kept at ambient conditions. Detection was carried out at 230 nm, achieving a resolution greater than 1.5 between L-DBTA and D-DBTA. The method demonstrated excellent linearity over a range of 30%-200% of the specification limit, with accuracy and range established from the LOQ level to 200%. Solution stability was confirmed for 1 day at room temperature, and precision was validated at both the LOQ and 100% levels. All validation parameters met the acceptance criteria, confirming the method's suitability for routine testing and batch release at quality control sites. This HPLC method is both sensitive and selective, ensuring the reliable determination of chiral purity in L-DBTA and its impurities.