The applicability of spectral analysis in detection of freshwater metal contamination was assessed by developing and testing a novel hyperspectral imaging (HSI) application for aquatic insect larvae (Trichoptera: Hydropsychidae). Larvae were first exposed to four different cadmium (Cd) concentrations: 0, 1, 10, and 100 μg L−1 for 96 h. Individual larvae were then preserved in ethanol, inspected with microscopy for the number of anomalies in larval gills, and imaged by hyperspectral camera operating with wavebands between 500 and 850 nm. Three additional larvae from each exposure were analyzed for tissue Cd concentration. Although the larval tissue Cd concentrations correlated positively with actual water concentrations, the toxicity response of larvae, i.e., frequency of gill abnormalities, did not differ among the Cd concentrations. In contrast, hyperspectral imaging data indicated some concentration-response relationship of larval spectral properties to the Cd exposure, but it was too weak for reliable automatic distinction between exposed and unexposed larvae. In this pilot study a workflow for data processing for a novel application of hyperspectral imaging was developed. Based on the results of this preliminary study, the workflow in the imaging process will be optimized and its potential for detecting metal contamination of aquatic environments reassessed.