Developing diagnostic tools based on the application of known disease/phenotype-associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user-friendly multiplex CNV detection method, termed stuffer-free MLPA-CE-SSCP, that combines a variation of multiplex ligation-dependent probe amplification (MLPA) with CE-SSCP. In this variation, MLPA probes were designed without the conventionally required stuffer sequences. To separate the similar-sized stuffer-free MLPA products, we adopted CE-SSCP rather than length-dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X-chromosome copy number (15) revealed that copy number determinations using stuffer-free MLPA-CE-SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation.