Vanadium Pentoxide (V2O5) nanoparticles exhibit diverse properties and have been studied for a wide range of applications, including energy storage, catalysis, environmental remediation, and material enhancement. In this work, we have reported the synthesis of Vanadium Pentaoxide (V2O5) nanoparticles using hydrothermal method. Ammonium metavanadate (NH4VO3) was used as a source of Vanadium. These syntheses were carried out at four different concentrations of Vanadium source. The hydrothermal reaction was conducted at a temperature of 180°C for a duration of 24 hours, followed by an additional 24-hour period of natural cooling. Four samples were annealed in air using a muffle furnace at 500 °C for five hours. The X-ray diffraction (XRD) technique was used to study the structural aspects. A comparative analysis of the microstructure was conducted utilizing the Scherrer method, the Williamson-Hall method and its various models, Size-Strain analysis, and the Halder-Wagner method. The crystallite size and microstrain were determined using these distinct methods, revealing a systematic correlation between the crystallite size and microstrain obtained through the different techniques.