Poplar species are economically important sources of timber and bioenergy and they also have a valuable application in phytoremediation and in forest restoration programmes. The characterization of their responses in the face of environmental constraints is essential to ascertain their adaptation capacity. Quantification of chloroplast pigments and antioxidant compounds, nutrients and carbon isotope discrimination (Δ13C) of a large set of samples in ecophysiological studies is important for determining the plant’s physiological responses. Δ13C and nitrogen are parameters of special interest for providing information on photosynthesis, water relations and nutritional characteristics in forest stands. Photoprotective and antioxidant compounds participate in the physiological defence of plants subjected to stressful environmental conditions. The measurement of these leaf components by traditional techniques is laborious and expensive. To evaluate them, a calibration model was obtained using Near-Infrared Reflectance Spectroscopy (NIRS) and chemical analyses of Populus spp. leaves sampled from different clones in different years under different physiological conditions. Predictive calibration equations for the concentration of chloroplast pigments (Chlorophylls a and b, lutein, neoxanthin, β-carotene (β-Car), VAZ (violaxanthin + antheraxanthin + zeaxanthin), epoxidation index (EI = (0.5·antheraxanthin + violaxanthin)/VAZ)), antioxidants (total phenolics (TPhe), ascorbate and α-tocopherol), Δ13C and N content were established using a partial least squares regression algorithm. Results showed a remarkable accuracy of the calibration equations for quantifying several antioxidative and photoprotective defence compounds such as neoxanthin (R2 = 0.844), β-Car (R2 = 0.827), VAZ (R2 = 0.846), EI (R2 = 0.831) and excellent accuracy for TPhe (R2 = 0.957), N (R2 = 0.963) and Δ13C (R2 = 0.922). As shown, results can be obtained on multiple physiological attributes with a single measurement, saving time and money and demonstrating the suitability of NIRS for ecophysiological purposes and bioremediation programs.