<p>In molecular diagnostics, the sequence-specific extraction and detection of nucleic acids remain a significant challenge. Here, we present a straightforward method that enables both the sequence-specific enrichment and subsequent analysis of nucleic acids using MALDI-TOF MS as a readout. Functionality and performance were tested successfully by using magnetic beads coated with DNA, which is complementary to the target nucleic acid, as a substrate. For sequence-specific extraction, hybridization of the target nucleic acid was performed, and subsequently, the single-stranded overhang was digested. The analysis was performed by <i>matrix-assisted laser desorption/ionization time-of-flight mass spectrometry</i> (MALDI-TOF MS) without further elution. The specificity was determined by hybridization of DNA oligonucleotides, which differ in up to four nucleobases in the middle of the strand. Two mutations led to a significant signal reduction. Mutated, but hybridizing strands can be distinguished from an entirely complementary strand by their mass. The specificity is higher for mutations located in the middle of the target strand. By employing single-strand-specific nucleases, we successfully digested a single-stranded overhang, resulting in the most intense signal corresponding to the blunt end. Knowing the mass of the immobilized oligonucleotide, the mass of the searched complementary mass can be calculated. Therefore, even with more than one digestion product, the searched oligonucleotide can be determined by its mass. The integration of hybridization selectivity, minimal sample preparation, short measurement time, and mass accuracy results in a robust and precise method for detecting specific sequences.</p> Graphical abstract <p></p>