Peptide nucleic acids (PNAs) are a new class of DNA mimics in which the regular nucleobases of adenine, thymine, cytosine, and guanine are connected via a peptide-like backbone. PNA molecules retain the same Watson-Crick base pairing as regular oligonucleotides, with the added benefits of greater specificity and resistance to enzymatic digestion. While the use of PNAs has grown rapidly because of their potential applications in biotechnology, little work has been done on developing analytical procedures for characterizing them. We have found matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to be an effective tool for PNA analysis. PNA molecules survive the MALDI process intact and are easily ionized with almost no multiply-charged species. These features allow mixtures to be easily characterized. Traditional protein matrices (e.g., sinapinic acid,2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid) were found to be superior to DNA matrices (e.g., trihydroxy-acetophenone, 3-hydroxypicolinic acid, picolinic acid). In addition, the new DNA matrix 6-aza-2-thiothymine worked well. The ability of MALDI-TOF-MS to ascertain PNA purity and sequence information at low picomole levels will be important as greater quality control of PNA synthesis is needed (e.g., when PNAs are used as antisense or antigene drugs).