In neonatal and pediatric intensive care units, Gram -positive infections are a significant cause of morbidity and mortality. The increase in infections caused by methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococci have led to the increased use of glycopeptides, which treat invasive infections caused by Gram -positive organisms, particularly those resistant to beta-lactam antibiotics. Teicoplanin has bacteriostatic activity against Gram -positive bacteria, but its pharmacokinetics in children is highly variable, with most children failing to reach target levels at the recommended dose. This study aimed to develop a cost-effective method for determining concentrations using dried blood spot (DBS). A method to determine the concentrations of teicoplanin in 20 µL blood or plasma using the Whatman 903 Protein Saver filter was evaluated. High-performance liquid chromatography with ultraviolet detection high-performance liquid chromatography with ultraviolet/vis was used, with internal standard ketoconazole. In addition, a method to quantify teicoplanin using 50 µL of liquid plasma was established to compare the results with the values obtained by DBS and dried plasma methods. The method was successfully developed and validated for 20 µL DBS. Furthermore, 50 µL of plasma was used to quantify teicoplanin with a lower limit of quantification of 10 mg/L. Precision and accuracy ranged from 2.3% to 10.7% and 95%-114.2%, respectively. A consistent factor (1.15) was used to calculate teicoplanin plasma concentrations from whole blood, indicating the reliability of the DBS method for therapeutic drug monitoring of teicoplanin. A simple, reliable, and cost-effective method using high-performance liquid chromatography with ultraviolet/vis was established to determine pediatric teicoplanin concentrations in both small plasma sample volumes and whole blood using DBS, and an accurate correlation factor for estimating teicoplanin plasma concentrations from DBS was identified. This method is suitable for the use in pediatrics.