We have successfully characterized the structure of testosterone, one of the essential steroids, through high-resolution rotational spectroscopy. A single conformer has been detected, and a total of 404 transitions have been fitted, allowing a precise determination of the rotational constants. It allowed us to unravel that the isolated structure of testosterone adopts an extended disposition. The results obtained in this work highlight how using laser ablation techniques in combination with Fourier transform microwave techniques allow the study of large biomolecules or common pharmaceuticals. It is an important step toward studying relevant biomolecules and developing new analytical techniques with unprecedented sensitivity and resolution.