Traditional duckweed toxicity tests only measure plant growth inhibition as an endpoint, with limited effects-based data. This study aimed to investigate whether Fourier-transform infrared (FTIR) spectroscopy could enhance the duckweed (Lemna minor L.) toxicity test. Four chemicals (Cu, Cd, atrazine, and acetochlor) and four metals-containing industrial wastewater samples were tested. After exposure of duckweed to the chemicals, standard toxicity endpoints (frond number and chlorophyll content) were determined; the fronds were also interrogated using FTIR spectroscopy under optimized test conditions. Biochemical alterations associated with each treatment were assessed and further analyzed by multivariate analysis. The results showed that comparable ECx (x percent of effective concentration) values could be achieved based on FTIR spectroscopy in comparison to those based on traditional toxicity endpoints. Biochemical alterations associated with different doses of toxicant were mainly attributed to lipid, protein, nucleic acids and carbohydrate structural changes, which helped explain toxic mechanisms. With the help of multivariate analysis, separation of clusters related to different exposure doses could be achieved. This is the first study showing successful application of FTIR spectroscopy in standard duckweed toxicity tests with biochemical alterations as new endpoints.