Latamoxef, a broad-spectrum anti-bacterial agent of the -lactam antibiotics, is used off-label in treatment of neonatal sepsis. Large inter-individual variability and uncertainty of treatment make therapeutic drug monitoring (TDM) useful to optimize antimicrobial therapy. The objective of this study was to develop and validate a simple, selective and reliable HPLC method for the determination of latamoxef in small volumes of plasma, which could be used in neonatal TDM. After a simple protein precipitation, analytes were separated with liquid chromatography and quantified by UV detection, with tinidazole as the internal standard. The calibration range was linear from 3.0 to 60.0g/mL. Intra- and inter-day precisions were<7.2%. The acceptance criteria of accuracy (between 85 and 115%, 120% for lower limit of quantification) were met in all cases. A plasma volume of 50L was required to achieve the limit of quantification of 3.0g/mL. The TDM results showed a large variability in trough concentrations. A large number of patients were underdosed, highlighting the unmet need for TDM to optimize latamoxef therapy in neonates.