Abstract Nucleic acid chips are based on the method of sequencing by hybridization, where unknown DNA fragments are hybridized to complementary nucleic acid sequences that are immobilized on a solid surface in an array format. One novel approach is to use peptide nucleic acid (PNA) biosensor chips. These DNA analogue possess the ability to hybridize with complementary DNA sequences. Because the backbone of DNA contains phosphates as opposed to PNA that does not, a technique that identifies the presence of these phosphates in a molecular surface layer would allow unlabelled DNA fragments hybridized to complementary PNAs to be detected. We have successfully shown that time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) is a very useful tool for identifying hybridized DNA on PNA biosensor chips by detecting the phosphorus present in the DNA. ToF‐SIMS is also a very effective technique for studying the complexity of the immobilization and hybridization process. Copyright © 2002 John Wiley & Sons, Ltd.