Neurochemical differential diagnosis between Alzheimer’s disease and other dementias is currently based on CSF biomarkers. Here, we report a method by which potential biomarkers can be identified in blood. Blood plasma samples from seven well-characterized Alzheimer’s patients and seven non-Alzheimer’s patients were subjected to a multi-dimensional protein separation procedure. After removal of 12 high-abundance proteins, the depleted samples from both diagnostic groups were labeled with different fluorescent dyes, mixed and separated by anion exchange and RP-chromatography. The resulting chromatography fractions were analyzed on 2D-gels. Twenty significant differentially expressed proteins were identified by mass spectrometry analysis. Ten of these proteins were either involved in the pathophysiology of Alzheimer’s disease or the amyloid/Aβ-peptide processing pathway. This work demonstrated a successful application of a multidimensional separation technique and holds the potential to identifying blood-based biomarkers for other diseases in the future.