The feasibility of proton minibeam radiation therapy (pMBRT) using a multislit collimator (MSC) and a scattering device was evaluated for clinical use at a clinical proton therapy facility. We fabricated, through Monte Carlo (MC) simulations, not only an MSC with a high peak-to-valley dose ratio (PVDR) at the entrance of the proton beam, to prevent radiation toxicity, but also a scattering device to modulate the PVDR in depth. The slit width and center-to-center distance of the diverging MSC were 2.5 mm and 5.0 mm at the large end, respectively, and its thickness and available field size were 100 mm and 76 × 77.5 mm², respectively. Spatially fractionated dose distributions were measured at various depths using radiochromic EBT3 films and also tested on bacterial cells. MC simulation showed that the thicker the MSC, the higher the PVDR at the phantom surface. Dosimetric evaluations showed that lateral dose profiles varied according to the scatterer’s thickness, and the depths satisfying PVDR = 1.1 moved toward the surface as their thickness increased. The response of the bacterial cells to the proton minibeams’ depth was also established, in a manner similar to the dosimetric pattern. Conclusively, these results strongly suggest that pMBRT can be implemented in clinical centers by using MSC and scatterers.