The introduction of high-throughput techniques is increasingly providing abundant information on molecular alterations requiring validation at the posttranscriptional level. Protein expression is now efficiently evaluated in large series of tumors included in tissue microarrays. We propose, describe and validate a technique to elaborate paraffin-embedded cell line microarrays (PECLIMA) from fixed cell cultures, which can be processed like standard surgical pathology biopsies prior to immunophenotyping. Our results show a reliable protein immunoexpression profiling in six widely used cell lines under different fixation conditions. This technique permits the simultaneous analysis of multiple antigens in multiple cell lines under different experimental conditions. Additional features of these arrays are long-term storage, their suitability for a variety of techniques including immunocytochemistry and in situ hybridization and their low cost.