Genomic deletions and duplications play an important role in the etiology of human disease. Versatile tests are required to detect these rearrangements, both in research and diagnostic settings. Multiplex ligation‐dependent probe amplification (MLPA) is such a technique, allowing the rapid and precise quantification of up to 40 sequences within a nucleic acid sample using a one‐tube assay. Current MLPA probe design, however, involves time‐consuming and costly steps for probe generation. To bypass these limitations we set out to use chemically synthesized oligonucleotide probes only. The inherent limitations of this approach are related to oligonucleotide length, and thus the number of probes that can be combined in one assay is also limited. This problem was tackled by designing a two‐color assay, combining two sets of probes, each amplified by primers labeled with a different fluorophore. In this way we successfully combined 28 probes in a single reaction. The assay designed was used to screen for the presence of deletions and duplications in patients with hereditary multiple exostoses (HME). Screening 18 patients without detectable point mutations in the EXT1 and EXT2 genes revealed five cases with deletions of one or more exons: four in EXT1 and one in EXT2. Our results show that a two‐color MLPA assay using only synthetic oligonucleotides provides an attractive alternative for probe design. The approach is especially suited for cases in which the number of patients to be tested is limited, making it financially unattractive to invest in cloning. Hum Mutat 24:86–92, 2004. © 2004 Wiley‐Liss, Inc.