Synchrotron X-ray diffraction data (SXRD) has been used to characterize the microstructural changes in 3.5 MeV proton irradiated 2.25Cr–1Mo (T22) steel samples as a function of dose through different techniques of X-ray diffraction line profile analysis (XRDLPA). Williamson-Hall (W–H) method, modified Rietveld method and convolutional multiple whole profile (CMWP) fitting technique have been used to evaluate the coherent domain size, r.m.s microstrain, dislocation density and dislocation character from SXRD data. It is demonstrated that XRDLPA from SXRD data is a powerful tool for extracting the microstructural parameters of irradiated samples in a reliable manner. Transmission Electron Microscopy (TEM) investigation has been carried out on unirradiated and irradiated samples to corroborate the results of XRDLPA. It is observed that dislocation density increases with dose and forms dislocation loops at highest dose. Vickers microhardness measurement has also been carried out to get the information on the mechanical property changes with dose. The variation in Vickers microhardness as a function of dose has been explained using the results from XRDLPA and TEM analysis.