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Mass spectrometry (MS) is an essential detection tool in bioanalytical chemistry owing to its exceptional selectivity and sensitivity, paired with rapid analyte identification and quantification. In this thesis, two classical MS and a newly developed ionization method are employed, including matrix-assisted laser desorption/ionization (MALDI) MS, electrospray ionization (ESI) MS and electrostatic spray ionization (ESTASI) MS. To meet the emerging challenges in bioanalytical chemistry, from disease diagnosis to drug development, new MS-based analytical methods ought to be developed with respect to high sensitivity, throughput, speed, as well as sample consumption and experimental simplification. For these purposes, this thesis presents four analytical strategies combined with MS detection for improved analytical performance in different research fields. Two classical analytical tools, thin layer chromatography (TLC) and 384-well plate, were coupled with ESTASI-MS respectively. In ESTASI-TLC-MS, ESTASI was applied to extract and identify a wide range of molecules of different polarities and chemical structures from both hydrophilic or hydrophobic silica plates with high sensitivity and minimal sample consumption in the femtomole range. In 384-well plate ESTASI-MS, the commercial 384-well plate could work as a container and an emitter for sample spray ionization, without any liquid delivery system or any additional interface. This approach provides fast and high throughput analyses for the large batches of reactions, such as enzyme assay and drug metabolism. Tyrosinase-catalyzed tyrosine oxidation in the presence or absence of inhibitors and cytochrome P450-catalyzed metabolic reactions of two drugs were studied respectively. To improve analysis sensitivity, two strategies, mass barcoded gold nanoparticles (Mb-AuNPs) for MALDI-MS signal amplification and microfabricated on-chip spyhole (Ø 10-12 ¿m) emitter for nanoelectrospray (spyhole-nanoESI), were developed, leading to a low sample consumption and high analysis efficiency. Mb-AuNPs combined with magnetic separation were applied for multiplex cow¿s milk allergy diagnosis in a component-resolved manner. IgE antibodies (Abs) could be extracted from a patient¿s blood serum by the formation of a sandwich structure between allergenic proteins-coated Mb-AuNPs and anti-human IgE Abs-functionalized magnetic beads (MBs). Detection of Mb-AuNPs by MALDI-MS provides a limit of detection (LOD) down to picograms per milliliter level for specific IgE Abs from only 1 ¿L of patient¿s blood serum. To take advantages of microfluidics in low sample consumption and easy integration, a novel interface of spyhole-nanoESI was designed for coupling microfluidics with MS and showed an improved sensitivity than standard ESI. This disposable microchip coupled with MS/MS shows potential application in cancer diagnosis by the successful detection of a small cell lung cancer biomarker in 1 ¿L of human serum at the extensive stage, without any complicated sample preparation steps.