This study was demonstrated to estimate the measurement uncertainty of 23 multiple-component amino acids from beef bone extract by high performance liquid chromatography (HPLC). The sources of measurement uncertainty (i.e. sample weight, final volume, standard weight, purity, standard solution, calibration curve, recovery and repeatability) in associated with the analysis of amino acids were evaluated. The estimation of uncertainty obtained on the GUM (Guide to the expression of uncertainty in measurement) and EURACHEM document with mathematical calculation and statistical analysis. The content of total amino acids from beef bone extract was 36.18 g/100 g and the expanded uncertainty by multiplying coverage factor (k, 2.05~2.36) was 3.81 g/100 g at a 95% confidence level. The major contributors to the measurement uncertainty were identified in the order of recovery and repeatability (25.2%), sample pretreatment (24.5%), calibration-curve (24.0%) and weight of the reference material (10.4%). Therefore, more careful experiments are required in these steps to reduce uncertainties of amino acids analysis with a better personal proficiency improvement.