Addressing the planetary crisis associated with climate change, biodiversity loss, global pollution, and public health requires novel and holistic approaches. Here, we present the methodology and initial results of an experiment conducted in Rome within the framework of the National Biodiversity Future Center (NBFC) project, Spoke 6. The major objective of this study was to outline the planetary health approach as a lens to assess urban health. This transdisciplinary case study explored the relationship between urban traffic-related external exposome and pro-oxidative responses in humans and plants. This methodology is based on the integration of atmospheric dynamics modeling, state-of-the-art aerosol measurements, biomonitoring in human cohorts, in vitro cellular assays, and the assessment of functional trait markers in urban trees. The results indicate that short-term exposure to urban aerosols, even at low concentrations, triggers rapid oxidative and inflammatory responses in bronchial epithelial cells, modulates gene and miRNA expression, alters gut microbiota diversity, and induces functional trait changes in urban trees. This study also highlights the feedback mechanisms between vegetation and atmospheric conditions, emphasizing the role of urban greenery in modulating microclimate and exposure. The methodology and initial results presented here will be further analyzed in future studies to explore proof of a cause–effect relationship between short-term exposure to traffic-related environmental stressors in urban areas and oxidative stress in humans and plants, with implications for chronic responses. In a highly urbanized world, this evidence could be pivotal in motivating the widespread implementation of planetary health approaches for assessing urban health.