Metabolomics aims at the comprehensive assessment of a wide range of endogenous metabolites and attempts to identify and quantify the attractive metabolites in a given biological sample. These metabolites have diverse physicochemical properties and are presented at different concentration ranges, which makes global analysis a difficult challenge. As a commonly used analytical platform, mass spectrometry (MS) coupled with various separation techniques, such as gas chromatography (GC) and liquid chromatography (LC), has recently undergone rapid development, providing promising solutions to these problems. The ambient ionization techniques, including desorption electrospray ionization (DESI), direct analysis in real time (DART) and extractive electrospray ionization (EESI), enable rapid detection of metabolites, making it ideal for high-throughput analysis in large-scale metabolomics studies. The current applications of these approaches are described with selected illustrative examples in the present review. Furthermore, regardless of “targeted” or “non-targeted” metabolomics study, the identification of the attractive biomarkers is required to further interpret the related metabolic pathways. Therefore, in the present review, recent novel MS-based techniques that allow more robust and easier metabolite identification are summarized, and their strengths and limitations are also discussed.