The performance of an original CE-MS interface that allows the in-axis positioning of the electrospray with respect to the MS inlet was evaluated. The variations in the geometrical alignment of this configu- ration in the absence of a nebulizing gas afforded a significant reduction in the sheath-liquid flow rate from 3 μL/min to as low as 300 nL/min. The sheath liquid and BGE were respectively composed of H2O—iPrOH–CH3COOH 50:50:1 (v/v/v) and 10% acetic acid (pH 2.2). A significant gain in sensitivity was ob- tained, and it was correlated to the effective mobility of the analytes. Compounds with low mobility val- ues showed a greater sensitivity gain. Special attention was paid to the detection of proteinogenic amino acids. Linear response functions were obtained from 15 ng/mL to 500 ng/mL. The limits of quantification, as low as 34.3 ng/mL, were improved by a factor of up to six compared to the conventional configu- ration. The in-axis setup was ultimately applied to the absolute quantification of four important amino acids, alanine, tyrosine, methionine and valine, in standard reference material (NIST plasma). The accura- cies ranged from 78 to 113%, thus demonstrating the potential of this configuration for metabolomics.