Abstract: Phospholipid analysis has become one of the research hotspots in lipidomics due to their important roles in signal transduction processes and functions of biological membrane. Matrix-assisted laser desorption-ionization mass spectrometry/imaging mass spectrometry (MALDI-MS/IMS) has been confirmed to be an advanced technique for profiling and imaging phospholipids(PLs). Optimization of matrix system and sample preparations according to the properties of PLs are critical steps for this technique. In order to establish a matrix system with high vacuum stability (stable more than 2 h), good tolerance to high frequency laser radiation, no matrix peak interference and uniform distribution, which does not cause the analyte displacement or migration, and can completely desorb and ionize different structures and concentrations of PLs to obtain accurate spatial distribution characterization of each phospholipid, many researchers had done a lot of work. Based on the development of the types of the matrix composition, progress in recent 10 years of matrix systems and sample preparation methods for MALDI-MS/IMS in profiling and imaging PLs was reviewed.