Large-scale screening to determine the mechanisms of anti-cancer actions of chemical libraries still presents technical challenges that are beyond the capabilities of conventional methods used in cellular or molecular biology. We recently demonstrated in a proof-of-concept study that infrared (IR) spectrum of cells exposed to anticancer drugs could be used to classify their mechanisms of actions. This study highlighted the fact that molecules inducing unique metabolic modifications could be selected for further pharmacological improvements. We show in this paper that drug concentration is an important parameter to be taken into account when analyzing mechanisms of anti-cancer actions by means of FTIR. The data indeed demonstrated that distinct spectral modifications occur in human PC-3 prostate cancer cells when exposed to ouabain at 10 × IC50 versus 1 × IC50. Longer incubation times at 1 × IC50 never resulted in spectral modifications fitting with those observed at 10 × IC50.