The American College of Medical Genetics recommends that each laboratory should confirm abnormal or ambiguous results detected by array comparative genomic hybridization (aCGH). At present, the gold standard method for aCGH confirmation is fluorescent in situ hybridization (FISH). However, FISH is not well suited for small tandem duplications or very small deletions that are detectable by oligonucleotide arrays. Therefore, we developed and validated multiplex ligation-dependent probe amplification (MLPA) for aCGH confirmation. The method performance validation showed linearity through the expected analytical measurement range (0.05 to 2 genome equivalents). The interassay normalized coefficient of variation averaged 3.7% across 12 control and target probes. This low imprecision allowed detection of 20% mosaicism with exceptional confidence (P<0.006). Comparison with a combined gold standard of phenotype, aCGH, karyotype, and/ or FISH showed 100% concordance for 218 samples using an X/Y chromosome-specific probe set (95% confidence interval, 98.3%-100.0%). Patient-specific probe sets also showed 100% concordance to the gold standard for 18 genomic targets. In conclusion, we have developed and validated an MLPA assay using a novel approach to accommodate the fact that positive controls would not be available at the time of testing. We initially validated the MLPA method using X/Y chromosome-specific probes and well-characterized samples and then validated new probe sets by comparision with reference populations. We have successfully incorporated aCGH confirmation using custom-designed MLPA into our normal workflow, and used it for confirmation of all abnormal or ambiguous results.