Nanotechnology is a transformative field due to its wide-ranging applications in biomedical, agricultural, and materials science. The unique physicochemical properties of nanoparticles, like high surface area and tunable size and shape, have emphasised their applications in targeted therapeutics, diagnostic devices, and improved nutrient delivery in crops. However, the increased application of nanoparticles has also raised concerns about toxicity, biodistribution, and environmental impact; thus, there is a need for analytical and regulatory approaches. Firstly, we have reviewed nanoparticles, their importance, and the toxicity. Following that, the chapter provides special emphasis on the role of X-ray-based techniques—X-ray diffraction (XRD) and X-ray fluorescence (XRF) techniques in characterising the structural parameters, particle size, crystallinity, phase composition, and adsorption behaviour in biological and plant materials. XRD and XRF are sophisticated tools that serve as an indispensable method for monitoring the interaction of nanoparticles at the molecular level, thereby advancing the effective application of nanoparticles.