Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) is an established tool for analyzing high mass molecules, such as proteins, whereas it attracts far less interest in the field of lipid analysis. In the study reported here a new chlorosulfolipid (CSL), 3,8,12,15-tetrachloroeicosane-1,17,18-triyl tris(hydrogen sulfate), was identified from the alga Ochromonas danica and de novo characterized by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-ToF) MS in negative ion mode. This method provides an effective alternative for the analysis of compounds directly derived from organic cell extracts. For MALDI analyses several frequently used solid MALDI matrices as well as some ionic liquid matrices (ILMs) were tested to enhance the analyte response to UV-laser and its ionization. The molecular weight of the observed compound could be determined as Li-, Na- and K-adducts [M+Me-2H]-. The characteristic isotopic patterns of the measured ions and the well-allocated molecular fragments by MS1, MS2 and MS3 indicate the fourfold chlorination and threefold sulfation of the investigated compound. The MS fragmentation alongside of the chlorine-bearing C-atoms is accompanied by the generation of a double bond at the opposite fragment in MS1. This obtained fragmentation pattern provides an insight into the allocation of the chlorine-bearing C-atoms along the carbon chain.