© 2019 Elsevier B.V. Single Particle Irradiation system to Cell (SPICE) facility at the National Institute of Radiological Sciences (NIRS) provides a magnetically focused 3.4 MeV proton microbeam. Throughout the radiobiological studies using SPICE over the years, we have realized that biological effect can be affected by the highly localized dose distribution of the microbeam and results may differ from that of broad beam irradiation. Previously, we have reported that the beam size of the SPICE microbeam was approximately 2 µm in diameter; the proton traversal was identified as etch pits produced on plastic nuclear track detectors, CR-39. However, this widely used method cannot distinguish individual proton tracks of highly focused microbeam due to the overlapping of etch pits, which was major limitation for beam size analysis as well as estimation of localized dose distribution in the cells. To overcome this limitation, we used fluorescent nuclear track detectors (FNTD; Al2O3: C, Mg) to investigate the beam size and its proton-number dependence (7–1000 protons per position). Detail analysis of Z(optical axis)-stacks of fluorescent images of the FNTD with a confocal laser scanning microscope clearly demonstrated that FNTDs has better spatial resolution and detection sensitivity compared to the conventional CR-39 detectors. The overall beam size was evaluated as elliptical shape or rectangular shape and determined to be in the range of 2.0 × 1.5–3.1 × 2.4 μm, depending on the number of protons per position. This result suggests that out-focused fraction of the incident protons contributes the broadening of the beam size. Throughout the present study, the FNTD irradiation and related image processing technology have demonstrated the feasibility of high-resolution measurement of the microbeam size over a wide range of the microbeam intensity.