The first step in an immunomonitoring strategy using ELISPOT or flow cytometry is to ensure the quality of fresh cells obtained from the patient. With the goal of standardizing the peripheral blood mononuclear cell separation process for the ELISPOT test at Thai Nguyen University of Medicine and Pharmacy, we conducted research on 50 blood samples from pregnant women infected with chronic hepatitis B. Using the cross-sectional descriptive method, we obtained the following results that the average age of pregnant women is 25.6 ± 3.9 years old. The percentage of living cells when separating fresh samples is 96% while in cold storage is 92.0%. Sample processing time is less than 12 hours, the percentage of living cells is 96.0% and the average concentration of PBMCs obtained is 2.9 x 106 (cells/mL). Cell quality is good when blood samples are separated as soon as possible after collection, ideally within 8–12 hours, of fresh samples.