Defensin (DF) is a new cytolytic antimicrobial peptide with a molecular weight of 4 kDa and antimicrobial activity against a wide range of gram positive and gram negative bacteria. The present work describes the development of the recombinant Defensin protein method using the internal standard bovine serum albumin (BSA). A simple, precise and accurate reversed-phase high-performance liquid chromatography method was developed and validated for the quantification of Defensin protein using BSA as an internal standard using the Enable Q C18 column (250 mm internal diameter x 4.6 mm, dimension of particles 5 μm 300ºA) as column, water : acetonitrile (60:40 v/v) with 0.1% trifluoroacetic acid as mobile phase, velocity flow rate of 1 ml/min and detection was performed at 280 nm. The retention time of BSA and Defensin was 2.342 and 5.279 minutes, respectively. The linearity range was found to be 10-50 μg/ml, the correlation coefficient was found to be 0.999. The limits of detection and quantification of the method were 1.010 and 3.062 μg/ml, respectively. Therefore, the developed method was accurate, precise and robust. Keywords: Recombinant defensin, BSA, RP-HPLC, validation. Human Defensins (1-5) are small, cationically charged, cysteine-rich endogenous antibiotic peptides with antimicrobial and cytotoxic properties that contain 29-35 amino acid residues, including six disulfide-linked invariant cysteine ​​moieties having a molecular weight of 4 -5 kDa. The new trend in drug development is the design and development of biopharmaceutical products, so the pharmaceutical industry has focused on the development and validation of biomolecule-based methods (8-9). HPLC is a widely used technique for proteins and peptides due to its ease of use, higher selectivity and faster analysis. In this study one of the antim...... middle of the paper ......Table 2). The results of the study on intraday (0.30 – 0.80%) and interday (0.34 – 0.62%) precision performed on 3 concentrations (20, 30 and 40 μg/ml) of BSA and Defensin standard solution indicate that the stated method is accurate (Table 2). The values ​​for LOD and LOQ were found to be 1.010 and 3.062 μg/ml, respectively (Table 2). These data suggested that the proposed method was sensitive for estimating both proteins. The recovery study was performed by the standard addition method. The amount of Defensin was calculated by inserting the value of the ratio between Defensin and BSA (IS) into the regression equation. And the recovery percentage was calculated for Defensin. The average recovery obtained was 99.82 – 101.85%, within the limit of 98 – 102%, which shows that the method was accurate (Table 3). To evaluate the robustness of the developed method, few parameters were deliberately varied