In order to test the plate reader’s sensitivity to treatment-induced changes in optical redox ratio, the cell lines were treated with the HER2 inhibitor trastuzumab for 24 h. As expected, the redox ratios of the HER2-overexpressing cell lines, SKBr3 and BT474, decreased with trastuzumab treatment (), while those of the HER2-negative cell lines, MCF7 and MDA-MB-231, did not change [Fig. 2(a)]. These trends match published multiphoton microscopy.13 To reaffirm the significant trends in redox ratios, NADH and FAD intensities were separately quantified for each group. As expected, the NADH and FAD autofluorescence intensities did not differ between the control and treated groups of HER2-negative cells [, Figs. 2(b) and 2(c)]. However, the fluorescence intensities of the SKBr3 and BT474 cells did exhibit significant changes (). Interestingly, the intensities of both NADH and FAD were higher in treated SKBr3 cells than the control, with a greater increase in FAD intensity leading to the decreased redox ratio. Conversely, the fluorescence intensities of both coenzymes in the treated BT474 cells decreased following the treatment; the overall decrease in redox ratio was caused by a greater decrease in NADH intensity than in that of FAD. Importantly, these differing trends in NADH and FAD autofluorescence intensities for each cell line agree with our previously published microscopy data of parallel experiments using BT474 cell monolayers,12,13 and highlight the utility of the redox ratio in measuring drug effect, rather than relying on trends shown by individual fluorophores. These results indicate that the metabolic sensitivity of the plate reader effectively measures changes in cellular metabolic state as an indicator of drug response. The evidence of these changes 24 h after treatment validates the instrument’s ability to quantify the optical redox ratio as accurately as microscopy methods with decreased equipment cost, data acquisition time, and number of samples. Quantification of this metabolic endpoint could be implemented in the early stages of the drug development process as a time- and cost-effective method to identify successful drug candidates.