The theses, lectures and solved problems within this book give an introduction to principles of mass spectrometry related to the analysis of biomolecules. The book focuses on MS and MS/MS analysis of peptides, oligosaccharides, lipids, glycoconjugates and polyphenols. The basic properties of modern MS ionization techniques and mass analyzers are presented, as well as appropriate MS methods for each class of biomolecules in modern biomedical scientific research. The biggest part of this book is focused on MS/MS analysis of biomolecules: how the MS and MS/MS data are generated, what are the basic strategies for their structural analysis, as well as how to interpret the respective MS and MS/MS data. This book also covers principles and examples of advanced MS/MS analyses, serving as a platform towards an intermediate level of understanding scientific research carried out in the field of structural MS analysis of biomolecules. It is the author’s hope that the reader will be able to understand principles of MS analyses related to their scientific and/or analytical interest, as well as design/help to propose MS and MS/MS experiments, as well as interpret the obtained data. This book is primarily written for students with basic knowledge of general, organic and analytical chemistry, and who are interested in applying MS-based methodologies in biomedical research fields, as well as interested in pursuing a career in mass spectrometry. Therefore it is best suited for senior undergraduate students and first/second year Ph.D. students. However, since MS instrumentation is rapidly adopted on all levels of scientific as well as analytical laboratories, the book should prove useful to scientists — as well as analysts — interested in applying MS in their upgraded laboratories. The author hopes that the reader, after studying the content presented, will be able to read primary scientific literature containing MS data to learn more about specific topics of interest, as well as be more capable of choosing a MS-related career path. For senior scientists, this book can offer some basic insights related to choosing a post-doc laboratory, or simply easier and more fruitful cooperation with mass spectrometrists.