We created double-stranded DNA (dsDNA) by annealing synthetic oligonucleotides and used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to measure the difference in molecular weight of the complementary strands after denaturation. Refined sample preparation and deliberate rectilinear construction at a linear mass spectrometer produced a mass resolution exceeding 1000 for single-stranded DNA. We present methodologies and demonstrate the potential for analysing dsDNA by determining the mass of the complementary strands.