The agricultural sector is required to produce food at the same pace as population growth, while accounting for pollution and costs. For this reason, conservative agricultural practices have been employed worldwide. Attenuated Total Reflectance–Fourier Transform Infrared (ATR-FTIR) spectroscopy has the ability to provide a snapshot of the macromolecular composition of a sample in a timely and cost-effective way and it has been widely applied in the field of agriculture to assess food quality. The aim of this study was to exploit ATR-FTIR spectroscopy to assess the impact of different soil tillage methods (conventional tillage, CT; minimum tillage, MT, and no tillage, NT) and nitrogen fertilization levels (0, 90 and 180 kg N ha−1) on the macromolecular composition of leaves and caryopses of durum wheat (Triticum turgidum subsp. durum). The analysis of the spectral data revealed that the quality of durum wheat, in terms of protein content, grown on soil with no tillage was not reduced. Indeed, with regards to caryopses, the different tillage methods influenced only the lipid and hemicellulose content, whereas the macromolecular composition of leaves was sensitive to tillage methods mostly during the early stage of growth. Moreover, no relevant effects were found in leaves and caryopses when different fertilizer concentrations were used. These results provide important knowledge supporting the adoption of both no-tillage soil treatments and reduced fertilization dosage for the development of durum wheat management strategies and support the use of spectroscopy for conservative agriculture practices.