Mass spectrometry (MS) has become the central technique that is extensively used for the analysis of molecular structures of unknown compounds in the gas phase. It manipulates the molecules by converting them into ions using various ionization sources. With high-resolution MS, accurate molecular weights (MW) of the intact molecular ions can be measured so that they can be assigned a molecular formula with high confidence. Furthermore, the application of tandem MS has enabled detailed structural characterization by breaking the intact molecular ions and protonated or deprotonated molecules into key fragment ions. This approach is not only used for the structural elucidation of small molecules (MW < 2000 Da), but also crucial biopolymers such as proteins and polypeptides; therefore, MS has been extensively used in multiomics studies for revealing the structures and functions of important biomolecules and their interactions with each other. The high sensitivity of MS has enabled the analysis of low-level analytes in complex matrices. It is also a versatile technique that can be coupled with separation techniques, including chromatography and ion mobility, and many other analytical instruments such as NMR. In this review, we aim to focus on the technical advances of MS-based structural elucidation methods over the past five years, and provide an overview of their applications in complex mixture analysis. We hope this review can be of interest for a wide range of audiences who may not have extensive experience in MS-based techniques.