Lactoferrin is increasingly supplemented in foods for its multiple functions. It is necessary to establish a reliable analytical method for nutritional assessment and quality control. A method for determination of native lactoferrin in milk using HiTrap Heparin HP column coupled with HPLC was developed and validated. Native lactoferrin was separated from denatured lactoferrin by centrifuging the samples that were adjusted to pH 4.6. Response surface design was used to find the optimal conditions for lactoferrin using HiTrap Heparin HP column as below: 10.35 mmol L⁻¹ disodium hydrogen phosphate for equilibration solutions, pH 6.02 for equilibration solutions, 1.78 mol L⁻¹ NaCl in elution solutions, and pH 7.26 for elution solutions. Subsequently lactoferrin was quantified using HPLC-PDA. A linear range from 2 to 100 mg L⁻¹ of lactoferrin standards was obtained, with a value of R² equal to 0.9989. The limit of detection (LOD) and quantification (LOQ) were 2.00 and 6.67 mg L⁻¹ for lactoferrin standard, and 0.57 and 1.90 mg L⁻¹ for milk sample considering enrichment factor involved in the pre-treatment procedures. In addition, milk spiked with lactoferrin at 3 concentration levels (2, 5 and 10 mg/L) showed that overall mean recovery were 88.3%, 90.2 and 95.1%, respectively. Relative standard deviation of intra-day and inter-day precision was 1.3–4.8%, and 2.1–5.7%, respectively, demonstrating good performances of the proposed method. The developed method was subsequently applied to determine lactoferrin in raw milk and processed milk. Results showed that this established method can be used to determine lactoferrin in different processed milk. Levels of lactoferrin in raw milk and processed milk were ranging from around 0.8 to 44.9 mg/L, indicating its level was strongly relying on the actual heat load that milk samples were exposed to. As expected, increasing the temperature from 72.5°C to 120°C resulted in lower native lactoferrin in milk. In addition, quantification of lactoferrin in raw milk and processed milk using this method displayed the usefulness and effectiveness of the proposed method.