||A new method is described for coupling electrokinetic chromatography (EKC) with mass spectrometry (MS) which allows the use of non-volatile, relatively large additives to achieve separation of analytes. This method involves the use of a novel, microdialysis membrane interface for EKC-MS. A membrane is used to selectively remove interfering additives while allowing transmission of analytes to the mass spectrometer where they are analyzed using electrospray MS. Two different designs were evaluated. The initial design, which placed the membrane within a liquid junction, did not prove to be satisfactory for optimal performance since a small positive inlet pressure was required to counteract the make-up flow back pressure within the junction. An improved design, which placed the membrane at the tip of the electrospray probe, did not have these limitations. This coaxial flow design was utilized for the demonstration of two EKC-MS applications. In the first application, the microdialysis membrane EKC-MS interface was utilized for the micellar electrokinetic chromatographic (MEKC)-MS separation of four β-blockers using a solution of a polyacrylic acid/polystyrene block copolymer. The separation was achieved in less than twenty minutes with partially resolved components easily distinguished using the mass selection available with MEKC-MS. The interface excluded the copolymer without affecting the retention of the β-blockers investigated. In the second application, capillary electrophoresis/frontal analysis-mass spectrometry (CE/FA-MS) was used for the determination of protein-drug binding. The microdialysis membrane EKC-MS interface enabled these analyses by selectively excluding the protein from introduction into the mass spectrometer, while allowing transmission of drug. The relative binding order for a series of eight β-blockers binding with α 1 -acid glycoprotein compared well with previously reported data from CE/FA-UV and conventional methods. The versatility of CE/FA analysis coupled with the sensitivity and universal detection possible with electrospray ionization-MS should allow the application of this technique to other protein-ligand systems. The membrane interface did not interfere with these measurements, by adding artifacts to the analyses. The application of the microdialysis membrane EKC-MS interface was quite successful. The interface was easily fabricated from commercially available components and was sufficiently rugged to allow several hundred experiments over the course of this work.