A laser microfabricated device was constructed for rapid microdialysis cleanup of biological samples for analysis by electrospray ionization mass spectrometry (ESI-MS) in both off-line and on-line modes. A microdialysis membrane was sandwiched between two chips having micromachined serpentine channels. The total volume of the sample serpentine channel used for the microdialysis was 1 microL. Efficient desalting was demonstrated for both DNA and protein samples using ESI with an ion trap mass spectrometer after microdialysis against a counter flow of ESI-compatible buffer. Signal-to-noise ratios were also greatly enhanced compared to direct infusion of the original nondialyzed samples. Importantly, the microfabricated device allowed use of sample flow rates 1 order of magnitude smaller than previous designs based on a microdialysis fiber, allowing reduced sample utilization and improved sensitivity with ESI-MS. The effectiveness of the cleanup was attributed to the size difference between the sample channel and the buffer channel and the fact that the sample is continuously refreshed by the buffer counterflow. The results indicate substantial potential for construction of highly compact and rugged devices enabling field applications of ESI-MS.