Tubulin is a heterodimeric protein composed of α- and β-subunits, which polymerize to form the cell’s microtubules. The latter are key components in mitotic spindle formation and essential targets in anticancer therapy. Compounds such as paclitaxel, tubulysins, dolastatins and synthetic analogues of these latter compounds, including cemadotin, exert their cytotoxic effects by disrupting microtubule dynamics. Previously, we reported the production and anticancer activity of a library of cemadotin analogues featuring a C-terminal tertiary amide functionalized with a variety of N-substituents, thus resulting in compounds occurring as a mixture of amide rotamers. Here we describe a comprehensive NMR and conformational study that provides new insights into the effect of the conformational equilibrium on the binding mode of the novel cemadotin analogues to the tubulin target. The conformational behavior of the isomer equilibrium of cemadotin’s terminal amide bond was investigated by TOCSY and ROESY NMR experiments, which allowed the identification and quantification of individual rotamer populations. A slow interconversion between the s-cis and s-trans amide rotamers was observed under standard NMR conditions (25 °C), indicating a significant energy barrier and conformational rigidity. Molecular docking and saturation transfer difference (STD) NMR experiments were performed with a representative analogue and tubulin to assess the binding mode. The results revealed that the s-trans rotamer is the predominant conformer in solution and exhibits a more favorable interaction with tubulin compared to the s-cis isomer, thus helping to understand the conformational requirements for an improved tubulin binding and the inhibition of the polymerization process.