Analysis of amino acid profiles in urine and plasma is an essential part of modern clinical diagnostic routine. Here we present an approach for the analysis of amino acids in urine by capillary electrophoresis/time-of-flight (TOF) mass spectrometry. At first a method combining improved separation, high dynamic range, and high sensitivity is presented. Detection limits in the mid nM-range are achieved through the use of pH-mediated stacking injection in combination with modern TOF detection technology. The method can be easily applied to detect differences in the amino acid profile in urine in a clinical context. Moreover, beside amino acids low molecular weight amines, peptides and related metabolites can be profiled. As a proof of concept, urine samples from patients suffering from osteoarthritis have been analyzed. Finally, the introduction of multivariate data analysis in the work flow was evaluated on spiked urine samples and real clinical material.