Abstract To accurately characterize the large amplitude motions and soft degrees of freedom of isolated molecules, sampling their conformational landscape by molecular mechanics and quantum chemical calculations may provide a valuable insight into the structure and dynamics. However, the resulting models need to be validated by a reliable experimental counterpart. For ethyl pentanoates, which belong to the family of fruit esters, benchmark calculations at different levels of theory showed that the C−C bond in proximity to the ester carbonyl group exhibits a large amplitude motion that is extremely sensitive to the choice of quantum chemical method and basis set. In such cases, insights from high‐resolution molecular jet techniques are ideal to accurately identify and characterize soft degrees of freedom. Here, we report on the most abundant conformer of ethyl 2‐ethyl butyrate using Fourier‐transform microwave spectroscopy. We show that – unlike other structurally related pentanoates for which gas‐phase and crystallographic data is available – ethyl 2‐ethyl butyrate possesses a C s symmetry plane under molecular jet conditions.