Frost damage remains an important driver of floral ecological dynamics in certain areas of the Australian landscape. However, responses of native Australian species to frost damage remain largely understudied. Here, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, conducted on intact leaves, was used to monitor changes in the protein secondary structures of Pittosporum spinescens upon exposure to below-zero temperatures. The dominant secondary structures present in fresh leaves were the inter-molecular aggregates (40 %), α-helices (20 %), β-sheets (15 %) and random coil structures (14 %). During simulated severe frost (-18 °C), a reduction in α-helices and increase in the amount of inter-molecular structures were observed, followed by transmutation of the latter into anti-parallel β-sheets or another form of inter-molecular structures. After 6 hours, the dominant protein secondary structures were anti-parallel β-sheets and inter-molecular aggregates (ca. 64 % and 17 %, respectively), with only small amounts of α-helices (4 %), β-sheets (9 %) and random coil structures (5 %) present. Overall, this indicates a reduction in the organisation level of protein secondary structures, resulting in a probable loss of function and considerable damage to the functional activity of any proteins in the leaves. The technique of ATR-FTIR spectroscopy should be considered by future researchers interested in investigating responses to frost damage in other species, particularly at an ecological level. Portable FTIR instrumentation would greatly expand the potential range of applications.