The utilization of graphene (G)-based materials e.g., graphene oxide (GO) and reduced GO (rGO) has significantly enhanced the field of mass spectrometry. G-based materials in question constitute a comprehensive collection that offers a wide range of applications, notably in the field of laser desorption/ionization mass spectrometry (LDI-MS). They enable the identification of many biological entities such as proteins, peptides, polysaccharides, and small compounds. G-based matrices offer high sensitivity. Graphene derivatives have been utilized in several advanced techniques, such as surface-assisted LDI-MS (SALDI-MS), surface-enhanced LDI-MS (SELDI-MS), and graphene-assisted LDI-MS (GALDI-MS). These methodologies demonstrate interferences-free spectra within the low mass range (50–1000 Da). They offered soft ionization MS and can be effectively utilized for the analysis of labile biomolecules.