Introduction: Within the last decade, the development of antigen microarray slides has enabled the simultaneous measurement of serum reactivity to hundreds of peptides in a single biological sample. Despite this considerable scientific progress, many issues remain regarding the quality, analysis and interpretation of the data these slides produce. There is currently no accepted approach to guide data analysis, and researchers use a wide variety of statistical methods and software tools. We designed and implemented a laboratory experiment to assess the reliability and range of measurement of peptide microarray data, and present graphical and statistical procedures for pre-processing so that quality data can be extracted for addressing biological hypotheses. Methods: Synthetic peptides spanning the proteins Ag85A, Ag85B, CFP10, MPT51/MPB51, TB10.4 and ESAT-6 were chosen as a paradigm to screen for serum reactivity to Mycobacteria tuberculosis (MTB). We explored various quantitative and graphical methods for presenting the responses from a slide. We replicated assays of samples from five TB-positive individuals to examine reproducibility, and used linear mixed models to investigate the various sources of variability, and to assess the range of measurement. We use our methods to extract data from the five TB-positive individuals and five healthy controls, and analyse the "normalized" responses using the freely available SAM package. Results: The ratio of foreground to background signal (on a log scale) provides an appropriate response index. A two-dimensional graphical display clearly illustrates the responses from the control and peptide features on a slide. Mixed model analysis of the replicated slides found a high reproducibility of the assay between operators, days and experiments. The range of measurement was also satisfactory. Our analysis of the normalized responses from the five TB-positive patients and five healthy controls suggested that 10 of the 363 peptides assessed had significantly higher responses in the TB-positive group. Conclusions: Carefully designed laboratory experiments and rigorous statistical analysis can enable the removal of technical artefacts to produce quality peptide array data for addressing biological hypotheses. These instruments, which enable valid Journal of Immunological Methods 328 (2007) 1 -13 www.elsevier.com/locate/jim Abbreviations: TB, tuberculosis; MTB, mycobacteria tuberculosis, M.tuberculosis (M.tub); MOTT, mycobacteria other than tuberculosis (MOTT); ESAT-6, early secreted antigenic target 6-kDa protein; FCS, fetal calf serum; FDR, false discovery rate; CFP10, culture filtrate protein 10; Ag85A, antigen 85A, a member of the Ag85 protein complex (Ag85A,-B, -C) comparisons across slides and/or batches of slides, will escort future comparative analyses targeting high content serum reactivity profiling against a broad array of B-cell epitopes.