The increasing use of engineered nanomaterials (ENMs) in agricultural practices offers promising benefits but also raises significant concerns about their interactions with plant systems and potential environmental impacts. This chapter provides an in-depth overview of advanced analytical methodologies that elucidate the localization, chemical speciation, and quantification of ENMs within plant tissues. Key techniques include electron and fluorescence microscopy, synchrotron-based X-ray spectroscopies, mass spectrometry, and single-cell analytical tools; together, these enable the investigation of nanoparticle uptake, translocation, biotransformation, and associated physiological responses. The integration of omics approaches and systems biology further provides a holistic framework for correlating the physical behavior of ENMs with molecular and cellular plant responses. The combined analytical strategies discussed here play a pivotal role in addressing the complexity of nanobiointeractions and in advancing the development of safe and effective nano-enabled agricultural technologies.