The combination of mass spectrometry, infrared action spectroscopy and quantum-chemical calculations provides a variety of approaches to the study of the structure of biologically relevant molecules in vacuo. This chapter reviews some of the experimental methods that are currently in use, which can roughly be divided into two main categories: (1) low-temperature neutral molecules in a molecular beam environment, which can be investigated in a conformationally selective manner by the application of double-resonance laser spectroscopy and (2) ionized species which can conveniently be manipulated and selected by mass spectrometric methods and which can be investigated spectroscopically by wavelength-dependent photo-dissociation. Both approaches rely on the application of infrared tunable laser spectroscopy and the laser sources most commonly used in current studies are briefly reviewed in Sect. 3. Along with quantum-chemical calculations, reviewed in Chapter 3 of this book (Gaigeot and Spezia, Top Curr Chem doi:10.1007/128_2014_620), the experimental IR spectra reveal a wealth of information on the structural properties of the biological species.