Abstract: Recent commercially available microfluidic chip-based nano-liquid chromatography system has greatly improved the robustness of nano-LC analysis. Chip-based nano-LC-tandem mass spectrometry (nano-LC-tandem MS) has been proven to enhance the resolution of LC separation and the sensitivity of MS detection. In this paper, we will discuss the utilization of chip-based nano-LC-tandem MS for the analyses of drug metabolite(7-aminoflunitrazepam), biomarker(8-isoprostane) and neurotransmitters in biological fluids for clinical diagnosis. The analytical performance(reproducibility, detection limit, linearity and precision) for various analytical compounds will be presented. In addition, matrix effect and ion suppression effect of human urine will be discussed. The applicability of microfluidic chip-based nano-LC-tandem MS for clinical diagnosis will be demonstrated.