Abstract The gastrointestinal disorder coeliac disease (CD) is induced by the ingestion of wheat gluten and is characterized by damage of the typical structure of the intestinal mucosa. The enzyme tissue transglutaminase (tTGase) was identified as the major target of disease-specific antibodies in-patients. We performed an epitope fine-mapping with a series of pentadecapeptides synthesized using parallel multiple peptide synthesis. For the detection of biomolecular interactions a label-free parallel method, reflectometric interference spectroscopy (RIfS), was used. This is the first optical label-free method adapted to a high throughput screening (HTS) format and the experimental results demonstrate its applicability as a biological screening device. A high titer of anti-tTGase antibodies is found in the serum of coeliac patients. We have taken the first step towards a fast non-surgical test for the detection of these antibodies. In order to identify and characterize a continous epitope with high affinity against the anti-tTGase antibody a screening of 21 pentadecapeptides has been accomplished with the parallel RIfS system. A single channel RIfS-system with high resolution was used to determine binding constants of identified peptides with high affinity.