DNA plasmids (pDNAs) are essential for gene cloning and protein expression, whereby engineered plasmids serve as vectors to insert foreign DNA into host cells, enabling mass production of proteins and vaccines. Due to the rapidly increasing use and application of a wide variety of pDNA (e.g., CRISPR-based gene editing, RNA therapeutics, and DNA vaccines), analytical methods to characterize their key attributes are vital. Here we explore mass photometry (MP) to analyze pDNAs and find that it completely fails using standard procedures as developed for MP on proteins, with masses underestimated by 30%-40%. While DNA can be measured by using coated glass slides, the large double-stranded DNA (dsDNA) particles diffract light beyond the diffraction limit, rendering most landing events unusable. To overcome such issues, we introduce a formic acid-based denaturation protocol to convert dsDNA particles rapidly (∼30 s) into single-stranded DNA (ssDNA)-like particles and show that these particles behave nearly perfect for MP. Using this protocol accurate and correct pDNA masses can be obtained, with values within 1-3% of the expected mass. Using this protocol, MP can be used to mass analyze pDNA constructs from 1 to 15 MDa, suggesting that this approach may be widely adopted within academia and biopharma for essentially all plasmids.