Biophysical and biochemical plant foliage parameters play a key role in assessing vegetation health. Those plant parameters determine the spectral reflectance and transmittance properties of vegetation; therefore, hyperspectral remote sensing, particularly imaging spectroscopy, can provide estimates of leaf and canopy chemical properties. Based on the relationship between spectral response and biochemical/biophysical properties of the leaves and canopies, the PROSPECT radiative transfer model simulates the interaction of light with leaves. In this study, more than 1100 leaf samples from the Amazon forest of Ecuador were collected at several study sites, some of which are affected by petroleum pollution, and across the vertical profile of the forest. For every sample, field spectroscopy at leaf level was conducted with a spectroradiometer. The goal of this study was to assess leaf optical properties of polluted and unpolluted rainforest canopies across the vertical profile and identify vegetation stress expressed in changes of biophysical and biochemical properties of vegetation. An ANOVA followed by Holme’s multiple comparisons of means and a principal component analysis showed that photosynthetic pigments, chlorophyll and carotenoids have significantly lower levels across the vertical profile of the forest, particularly in sites affected by petroleum pollution. On the other hand, foliar water content showed significantly higher levels in the polluted site. Those findings are symptoms of vegetation stress caused by reduced photosynthetic activity and consequently decreased transpiration and water-use efficiency of the plants. Cross-comparison between SPAD-502 chlorophyll content meter index and chlorophyll content showed strong positive correlation coefficients (r = 0.71 and r2 = 0.51) which suggests that using the SPAD-502 chlorophyll index itself is sensitive enough to detect vegetation stress in a multispecies tropical forest. Therefore, the SPAD-502 can be used to assess the chlorophyll content of vegetation across polluted and non-polluted sites at different canopy layers. The results presented in this paper contribute to the very limited literature on field spectroscopy and radiative transfer models applied to the vertical profile of the Amazon forest.