Non-aqueous capillary electrophoresis (NACE) is an attractive CE mode, in which water solvent of the background electrolyte (BGE) is replaced by organic solvent or by a mixture of organic solvents. This substitution alters several parameters, such as the pKa, permittivity, viscosity, zeta potential, and conductivity, resulting in a modification of CE separation performance (i.e., selectivity and/or efficiency). In addition, the use of NACE is particularly well adapted to ESI-MS due to the high volatility of solvents and the low currents that are generated. Organic solvents reduce the number of side electrochemical reactions at the ESI tip, thereby allowing the stabilization of the ESI current and a decrease in background noise. All these features make NACE an interesting alternative to the aqueous capillary zone electrophoresis (CZE) mode, especially in combination with mass spectrometry (MS) detection. The aim of this work was to evaluate the use of NACE coupled to negative ESI-MS for the analysis of acidic compounds with two available CE-MS interfaces (sheath liquid and sheathless). First, NACE was compared to aqueous CZE for the analysis of several pharmaceutical acidic compounds (non-steroidal anti-inflammatory drugs, NSAIDs). Then, the separation performance and the sensitivity achieved by both interfaces were evaluated, as were the impact of the BGE and the sample composition. Finally, analyses of glucuronides in urine samples subjected to a minimal sample pre-treatment (“dilute-and-shoot”) were performed by NACE-ESI-MS, and the matrix effect was evaluated. A 20- to 100-fold improvement in sensitivity was achieved using the NACE mode in combination with the sheathless interface and no matrix effect was observed regardless of the interfaces.