Understanding how organisms respond to trace elements is important because some are essential for normal bodily homeostasis, but can additionally be toxic at high concentrations. The inflection point for many of these elements is unknown and requires sensitive techniques capable of detecting subtle cellular changes as well as cytotoxic alterations. In this study, we treated human cells with arsenic (As), copper or selenium (Se) in a dose–response manner and used attenuated total reflection Fourier-transform infrared (ATR-FTIR) microspectroscopy combined with computational analysis to examine cellular alterations. Cell cultures were treated with Asv, Cu2+ or Seiv at concentrations ranging from 0.001 mg L−1 to 1000 mg L−1 and their effects were spectrochemically determined. Results show that Asv and Cu2+ induce bimodal dose–response effects on cells; this is in line with hormesis-driven responses. Lipids and proteins seem to be the main cell targets for all the elements tested; however, each compound produced a unique fingerprint of effect. Spectral biomarkers indicate that all test agents generate reactive oxygen species (ROS), which could either stimulate repair mechanisms or induce damage in cells.