Clustered DNA damage is considered as a critical type of lesions induced by ionizing radiation, which can be converted into the fatal or strong mutagenic complex double strand breaks (DSBs) during damage processing in the cells. The new data show that high energy protons produce more potentially lethal DSBs than low LET radiation. In this study, plasmid DNA were used to investigate and re-evaluate the biological effects induced by the protons with the LET of ∼3.6 keV/μm at the molecular level in vitro, including single strand breaks (SSBs), DSBs, isolated and clustered base damages. The results of complex DNA damage detections indicated that protons at the given LET value induce about 1.6 fold more non-DSB clustered DNA damages than the prompt DSB. The DNA damage yields by protons were greater than that by γ-rays, specifically by 6 fold for the isolated type of DNA damage and 14 fold for the clustered damage. Furthermore, the spectrum of damages was also demonstrated to be depended on the radiation quality, with protons producing more DSBs relative to clusters than do γ-rays.