Needle biopsies are taken as standard diagnostic specimens for many cancers, but no technique exists for the high-throughput analysis of multiple individual immunohistochemical (IHC) markers using these samples. Here we present a simple and highly reliable technique for constructing tissue microarrays (TMAs) from prostatic needle biopsies. Serial sectioning of the TMAs, called 'Checkerboard TMAs', facilitated expression analysis of multiple proteins using IHC markers. In total, 100% of the analysed biopsies within the TMA both preserved their antigenicity and maintained their morphology. Checkerboard TMAs will allow the use of needle biopsies (i) alongside other tissue specimens (trans-urethral resection of prostates and prostatectomies in the case of prostate cancer) in clinical correlation studies when searching for new prognostic markers, and (ii) in a diagnostic context for assessing expression of multiple proteins in cancers from patients prior to treatment.