Non-aqueous capillary electrophoresis (NACE) on microfluidic chips is still a comparatively little explored area, despite the inherent advantages of this technique and its application potential for, in particular, lipophilic compounds. A main reason is probably the fact that implementation of NACE on microchips largely precluded the use of polymeric substrate materials. Here, we report non-aqueous electrophoresis on a thiol-ene-based microfluidic chip coupled to mass spectrometry via an on-chip ESI interface. Microchips with an integrated ESI emitter were fabricated using a double-molding approach. The durability of thiol-ene, when exposed to different organic solvents, was investigated with respect to swelling and decomposition of the polymer. Thiol-ene exhibited good stability against organic solvents such as methanol, ethanol, N-methylformamide, and formamide, which allows for a wide range of background electrolyte compositions. The integrated ESI emitter provided a stable spray with RSD% of the ESI signal ≤8%. Separation efficiency of the developed microchip electrophoresis system in different non-aqueous buffer solutions was tested with a mixture of several drugs of abuse. Ethanol- and methanol-based buffers provided comparable high theoretical plate numbers (≈ 6.6 × 104–1.6 × 105 m−1) with ethanol exhibiting the best separation efficiency. Direct coupling of non-aqueous electrophoresis to mass spectrometry allowed for fast analysis of hydrophobic compounds in the range of 0.1–5 μg mL−1 and 0.2–10 μg mL−1 and very good sensitivities (LOD ≈ 0.06–0.28 μg mL−1; LOQ ≈ 0.20–0.90 μg mL−1). The novel combination of non-aqueous CE on a microfluidic thiol-ene device and ESI-MS provides a mass-producible and highly versatile system for the analysis of, in particular, lipophilic compounds in a wide range of organic solvents. This offers promising potential for future applications in forensic, clinical, and environmental analysis.