Current proteomic strategies depend strongly on the development of analytical methodologies and instrumentation. In parallel to the development of mass spectrometry (MS) - based proteomic workflows, microfluidic devices emerged in this field as a flexible tool for rapid and sensitive protein studies. In this context, the present work focuses on the development of miniaturized analytical systems for protein studies, especially by electrospray ionization mass spectrometric detection. Several approaches have been proposed to complement the mass spectrometric analysis of tryptic peptides using chemical tagging to isolate specific subclasses of proteins by affinity baits or for quantitation purposes. Optimization of a chemical tagging reaction in a sandwich mixer-reactor that consists of mixing two solutions providing the reactants in a channel, one central laminar flow being sandwiched between two outer flow solutions, was first investigated by finite-element method. The numerical simulations have highlighted the importance of positioning the reactant presenting the lowest diffusion coefficient close to the sidewall, as well as decreasing the outer flow velocities, to enhance a chemical reaction because of the parabolic flow profile across the microchannel. This optimization is even more relevant for consecutive reactions. As infusion-based electrospray microchips are known to present great advantages in terms of sensitivity compared to standard ionization source, an ESI emitter microchip based on polymer photo-ablation was designed. First, the hyphenation of the chip in a LC-MS workflow was successfully achieved and evaluated by comparison with a standard pneumatically-assisted ESI source. To perform enhanced on-chip post-column derivatization of peptides, a substitute design to the sandwich mixer-reactor was explored to improve reaction in a microchip before ESI – MS analysis. The electrospray micromixer chip includes a passive mixing unit to perturbate the flow along the microchannel. The mixing efficiency was demonstrated by fluorescence imaging, and on-chip chemical derivatization of peptides and kinetic studies before mass spectrometry were achieved. The on-line LC-MS derivatization of cysteinyl peptides was shown to provide a more confident and accurate protein identification by adding information on the peptidic sequence. Within the development of electrospray microchips for mass spectrometry, many efforts have been put forward in the hyphenation of functional units. Immobilization of functionalized stationary phase in microfluidic systems is widely used to achieve protein or peptide isolation, sample cleaning, separation or reaction. In this context, magnetic beads have proven to be quite useful compared to other methods, such as immobilized packed beads or monoliths, because of their high and reversible magnetization, and various surface functionalization. A polymer microchip including a magnetic track array was designed to focus the magnetic field generated by permanent magnets towards precise locations along the microchannel. A multi-plug magnetic bead capture was obtained and a significant increase of bead capture efficiency was demonstrated both numerically and experimentally, which is beneficial for affinity separation applications, as example. The research in microfluidic front-end devices for mass spectrometry is actually oriented to the development of multi-spray interfaces for high-throughput analysis. An electrospray microchip with a multi-track array was developed. The capability of the device to screen alternatively up to six samples and to perform relative quantification was assessed. Soft ionization techniques have revolutionized the analysis of proteins by mass spectrometry. In contrast with the major content of the thesis dealing with microfluidic systems for protein analysis, the last chapter presents a novel ambient ionization method, so-called membrane-desorption electrospray ionization (M-DESI). The principle is similar to the desorption electrospray ionization (DESI) method. But, in the M-DESI approach the sample is desorbed directly from a mesh membrane positioned vertically and in-axis between the ESI emitter and the mass spectrometer inlet. Analysis of peptides and proteins was demonstrated as a proof-of-concept, offering great perspectives for detection after separation on membrane.