Abstract: Protein characterization highly depends on its operating conditions, where native or often referred as non-denaturing options will greatly facilitate the collection of more biologically meaningful information. Native mass spectrometry (MS) is such a technique that enables protein analysis under non-denaturing conditions with an affordable time and sample cost. The indispensable roles of native MS-based strategies in the deep dissection of protein conformations, non-covalent interactions, and self-assembly stoichiometry have been seen in past decade, which suggest that native MS has increasingly grown to be a ground technique for the structural biology realm. In this review, we firstly introduced the current method development for protein extraction compatible with native MS experiments, ranging from traditional methods to in situ extraction means, which is key to achieve biologically meaningful native MS results. Then, we focused on the most recent advancements in the native MS-compatible ion source and novel regimes for protein conformational interpretation. Specifically, except the most frequently used nanoelectrospray ion source for native MS, we also listed a variety of emerging ionization regimes including nano-desorption electrospray ionization, submicron emitter tip electrospray, induced nanoelectrospray ionization, step-voltage nanoelectrospray ionization. For protein structural analysis, native MS relies on both charge state distribution and conformational interrogation tool, e.g. ion mobility spectrometry. The structural information can be further enriched when coupled to collision-induced unfolding technique, which was also discussed with most recent developments in data acquisition and visualization methods. Current challenges and potential solutions facing native MS realm in terms of both sampling methods and structural resolution were tentatively discussed.