Analytical approaches for sensing cellular NADH conformation from autofluorescence signals have significance because NADH is a metabolic indicator and endogenous biomarker. Recently, spectral detection of multiple cellular NADH forms during chemically-induced metabolic response was reported, however because NADH is solvatochromic and the spectral change is small, the possibility of a non-metabolic interpretation needs to be considered. Here we investigate the response of UV-excited autofluorescence to a range of well-known chemicals affecting fermentation, respiration, and oxidative-stress pathways in Saccharomyces cerevisiae. The two-component nature of the spectral response is assessed using phasor analysis. By considering a series of physically similar and dissimilar chemicals acting on multiple pathways, we show how the two-component nature of a spectral response is of metabolic origin, indicative of whether a single or several pathways have been affected.