Sweat is a body fluid that can be collected noninvasively. Sweat can be utilized as a biofluid for quantification of amino acids for clinical applications, such as disease screening. Amino acids are essential for many biological processes in the human body. Hence, the development of analytical methods for determining the concentrations of amino acids in human sweat can offer valuable insights into the health status of an individual. Sweat analysis can further be utilized as a tool for screening various pathological conditions. In this study, the concentrations of amino acids in small volumes of human sweat collected from fingertips of healthy subjects at rest were investigated. Reliable sweat collection was achieved by optimizing the sample collection procedure. The amino acids in human sweat were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, followed by analysis using high-performance liquid chromatography with fluorescence detection. The amino acid concentrations in sweat were quantified. The temporal variability and sex-specific differences in the concentrations of amino acids were evaluated. The results of both intra- and inter-day sweat analyses revealed that except for citrulline and arginine, the concentrations of other amino acids in sweat are relatively stable. This study proposes that the concentrations of amino acids in sweat can be determined with high accuracy, regardless of sweat collection timing. The findings of this study provide useful information that can help develop disease screening methods based on sweat analysis.