Thin Layer Chromatography (TLC) coupled to Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI-MS) offers the ability to couple a rapid and versatile separation technique with routine MALDI MS analysis providing an effective strategy for the analysis of lipid mixtures. Due to the varying detectability’s of individual lipid classes when using a MALDI Imaging Mass Spectrometry (MALDI-IMS) approach, prior separation using TLC offers advantages including detection of either low abundance or suppressed ions coupled with the ability to analyse multiple samples (e.g. control and infected) at the same time. The complement of lipids extracted from excised midguts of control and mosquitoes infected with GFP labelled Plasmodium berghei ANKA, were separated by TLC using optimised separation and matrix application conditions prior to MALDI Fourier Transform MS (FTMS) analysis. Using this approach we putatively identified a phosphatidylethanolamine (PE(38:5)) as a marker of the infection status. Subsequent tissue imaging using MALDI-FTMS and classification using Receiver Operating Characteristic (ROC) curve analysis of control and infected mosquitoes tissue sections confirmed the PE(38:5) to be collocated with the site of infection within the midgut. Further, a number of polyunsaturated fatty acids corresponding to eicosapentaenoic acid and eicosatetraenoic acid, implicated in both parasite development and host immune response, were found to be highly abundant in infected tissues sections.