Aminoacids and their derivatives are key biologically important metabolites and reliable, rapid and accurate, quantification for these analytes in urine remains an important analytical challenge. Here a fast and reliable HILIC-tandem MS method is presented for application in clinical or nutritional studies. The developed method was validated according to existing guidelines adapted for endogenous analytes. The validation strategy provided evidence of linearity, LOD and LOQ, accuracy, precision, matrix effect and recovery. The surrogate matrix approach was applied for calibration proving satisfactory accuracy and precision based on standard criteria over the working concentration ranges. Intra and inter day accuracy was found to range between 0.8 and 20% for the LQC (low QC) and between 0.05 and 15 % for MQC (medium QC) and HQC (high QC). Inter and intraday precision were found to be between 3 and 20 % for the LQC and between 1 and 15% for the MQC and HQC. The stability of the analytes, in both surrogate and pooled urine QC samples, was found to be within 15% over a short period at 4 °C or after a up to 3 freeze-thaw cycles. The uncertainty of the method was also assessed to provide increased confidence for the acquired measurements. The method was successfully applied to a subset of human urine samples involved in a study of amino acids dietary uptake. This method may provide a valuable tool for many applications or studies where amino acid metabolic signatures in the excreted urine are under investigation.