Appropriate methods for the analysis of microdissected solid tumour tissues by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing approximately 200 000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI-TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high-performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four-fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI-TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.