This lecture notes book presents how enhanced structural information of biomolecular ions can be obtained from interaction with photons of specific frequency - laser light. The methods described in the book 'Laser photodissociation and spectroscopy of mass-separated biomolecular ions' make use of the fact that the discrete energy and fast time scale of photoexcitation can provide more control in ion activation. This activation is the crucial process producing structure-informative product ions that cannot be generated with more conventional heating methods, such as collisional activation. The book describes how the powerful separation capabilities and sensitivity of mass spectrometry (MS) can be combined with the structural insights from spectroscopy by measuring vibrational and electronic spectra of trapped analytes. The implementation of laser-based photodissociation techniques in MS requires basic knowledge of tunable light sources and ion trapping devices. This book introduces the reader to key concepts and approaches in molecular spectroscopy, and the light sources and ion traps employed in such experiments. The power of the methods is demonstrated by spectroscopic interrogation of a range of important biomolecular systems, including peptides, proteins, and saccharides, with laser light in the ultraviolet-visible, and infrared range. The book 'Laser photodissociation and spectroscopy of mass-separated biomolecular ions' is an indispensable resource for students and researchers engaged or interested in this emerging field. It provides the solid background of key concepts and technologies for the measurements, discusses state-of-the-art experiments, and provides an outlook on future developments and applications.