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Amoxicillin, a broad-spectrum β-lactam antibiotic, is widely used for treatment of neonatal infections. Despite the unmet need in neonates, an adapted analytical method is still missing in clinical practice. The purpose of this study was to develop and test an easy and credible high performance liquid chromatography-UV (HPLC-UV) method to determine amoxicillin in small volumes of human plasma and use it in routine therapeutic drug monitoring (TDM) of neonates. After a protein precipitation, amoxicillin for standards, quality control samples, and patient samples were separated by LC and measured by UV detection, and tinidazole was used as the internal standard. The calibration range was 0.50-20.0 μg/mL. Intra- and inter-day precisions were less than 4.7%. The acceptance criteria of accuracy (between 85-115%) were met in all cases. A plasma volume of 50 μL was required to achieve the limit of quantification of 0.50 μg/mL. Thus, a simple, rapid, and accurate HPLC-UV method has been developed to detect the concentration of amoxicillin in human plasma. This method was adapted to do TDM of amoxicillin in neonates. [ABSTRACT FROM AUTHOR]