cDNA subtractive hybridization is a very powerful method to compare gene expression profiles between two cell or tissue samples of interest. The sample in which differentially expressed transcripts are to be found is usually referred to as “tester,” whereas the reference sample is called “driver.” A cDNA subtraction experiment should always be performed in both directions, with each sample serving as the tester and the driver in separate reactions. The general requirements to perform a cDNA subtraction and cloning procedure encompass the isolation of total RNA or mRNA from the specimens of interest, followed by reverse transcription of the mRNA into cDNA. The concept of cDNA subtraction is based on the hybridization of reverse-transcribed mRNAs present in both experimental samples. These sequences can form double-stranded hybrids that are then removed from the reaction. Only cDNAs from the tester population that remain single-stranded are further amplified, thus representing the pool of differentially expressed genes. This pool enriched for differentially expressed sequences can easily be cloned to generate a subtraction library. The analysis of subtraction efficiency is a crucial step after the procedure in order to rely on the results obtained. The subtractive hybridization is very valuable to achieve insights into genes differentially regulated between two samples of interest. Importantly, however, the generated cDNA pools enriched for genes overexpressed in either of the two tissue or cell specimens are also excellent probes for array hybridization applications—timely methods to identify gene expression changes on a large scale.