Objectives Copy number alterations (CNAs) are frequently found in malignant tissues. Different approaches have been used for CNA detection. However, it is not easy to detect a large panel of CNA targets in heterogenous tumors. Methods We have developed a CNAs detection approach through quantitatively analyzed allelic imbalance by allelotyping single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the copy number changes were quantified by real-competitive PCR (rcPCR) to distinguish loss of heterozygosity (LOH) and genomic amplification. The approach was used to validate the CNA regions detected by next generation sequencing (NGS) in early-stage lung carcinoma. Results CNAs were detected in heterogeneous DNA samples where tumor DNA is present at only 10% through the SNP based allelotyping. In addition, two different types of CNAs (loss of heterozygosity and chromosome amplification) were able to be distinguished quantitatively by rcPCR. Validation on a total of 41 SNPs from the selected CNA regions showed that copy number changes did occur, and the tissues from early-stage lung carcinoma were distinguished from normal. Conclusions CNA detection by MALDI-TOF MS can be used for validating potentially interesting genomic regions identified from next generation sequencing, and for detecting CNAs in tumor tissues consisting of a mixture of neoplastic and normal cells.