This work demonstrates the synthesis and application of in-situ synthetic peptide microarrays for a high-throughput parallel study of peptide-antibody interactions at amino acid level. A combination of solid phase peptide synthesis, photogenerated reagent chemistry and fluorescence imaging techniques was used to generate spatially addressable peptide microarrays with up to 12mer sequences. The synthesis substrate was a microfluidic microarray of individual reactors connected by channels, converging into a solution inlet and outlet, etched on a flat silicon surface sealed by a glass cover. A constant synthesis efficiency of >98% was observed for sequences of up to 9 amino acids. Peptide sequences from Respiratory Synctial Virus (RSV) were synthesized and used to identify binding sites to the antibody MAb 19 by performing on-chip, high throughput ELISA. Key residues for binding were identified by mutational and deletional analysis on these sequences. To further elucidate the biological applications of the synthetic peptides, the on-chip binding specificity of a 8mer peptide to a murine B lymphoma cell line was demonstrated.