The evaluation of microRNA profiles has represented one of the first approaches to investigate the aberrant microRNA expression in different human cancers, providing the first experimental evidence of the involvement of these small noncoding RNAs in the tumorigenic process. Currently, these and other methods have been applied to the search of new molecular biomarkers of diagnosis, prognosis, and response to therapy prediction. Here, we described the approach we used for the determination of a miRNA signature unique for human ovarian cancer, first performing a large-scale screening using a custom-made microarray platform, and then validating the obtained data by Northern blot or real-time PCR.