Radiofrequency fields in the 1–28 GHz range are ubiquitous in the modern world, giving rise to numerous studies of potential health risks such as cancer, neurological conditions, reproductive risks and electromagnetic hypersensitivity. However, results are inconsistent due to a lack of precision in exposure conditions and vastly differing experimental models, whereas measured RF effects are often indirect and occur over many hours or even days. Here, we present a simplified RF exposure protocol providing a single 1.8 GHz carrier frequency to human HEK293 cell monolayer cultures. A custom-built exposure box and antenna maintained in a fully shielded anechoic chamber emits discrete RF signals which can be precisely characterized and modelled. The chosen amplitudes are non-thermal and fall within the range of modern telecommunication devices. A critical feature of the protocol is that cell cultures are exposed to only a single, short (15 min) RF exposure period, followed by detection of immediate, rapid changes in gene expression. In this way, we show that modulation of genes implicated in oxidative stress and ROS signaling is among the earliest cellular responses to RF exposure. Moreover, these genes respond in complex ways to varying RF signal amplitudes consistent with a hormetic, receptor-driven biological mechanism. We conclude that induction of mild cellular stress and reactive oxygen species (ROS) is a primary response of human cells to RF signals, and that these responses occur at RF signal amplitudes within the range of normal telecommunications devices. We suggest that this method may help provide a guideline for greater reliability and reproducibility of research results between labs, and thereby help resolve existing controversy on underlying mechanisms and outcomes of RF exposure in the general population.