The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim of the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs. A novel design of MLPA probes has been developed to permit for MLPA analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A total number of 792 ligation events were analyzed. In DNA extracted from dried blood spot samples, 99.1% of the results were accurate compared to 99.9% of the results obtained in DNA from whole blood samples. This study clearly demonstrates that MLPA reactions with modified probes are successful and reliable with DNA concentrations down to 0.3 ng/microL (1.6 ng total). This broadens the diagnostic perspectives of samples of DBSS allowing for copy number variation analysis in general and particularly testing for CAH.