Recent advances in DNA microarray technology have enabled researchers to comprehensively characterize the complex genomes of higher eukaryotic organisms at an unprecedented level of detail. Array-based comparative genomic hybridization (Array-CGH) has been widely used for detecting DNA copy number alterations on a genomic scale, where the mapping resolution is limited only by the number of probes on the DNA microarray. In this chapter, we present a validated protocol utilizing print-tip spotted HEEBO (Human Exonic Evidence Based Oligonucleotide) microarrays for conducting array-CGH using as little as 25 ng of genomic DNA from a wide variety of sources, including cultured cell lines and clinical specimens, with high spatial resolution and array-to-array reproducibility.