A freezing technique protocol was proposed for coupling microchip electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The microfluidic flow was frozen immediately after electrophoresis on microfluidic chip and the separated analyte molecules were kept in their zone pattern in the electrophoresis. Then, the frozen-chip was lyophilized and sent into TOF-MS instrument as a MALDI target, and the analyte molecules in the microfluidic channels were subjected to analysis by mass spectrometry. This approach could eliminate sample cross-contamination, providing a new interface for microchip electrophoresis and MALDI-MS.