Abstract  I. Introduction 17  II. Techniques and Methods 18 A. Time‐of‐Flight Mass Spectrometer 18 1. Linear TOF‐MS 18 2. Reflectron 18 B. Resonance Enhanced Multi‐Photon Ionization (REMPI) 18 C. Threshold Ionization 20 1. PIPECO 20 2. Pulsed‐Field Threshold Ionization 20 3. Mass‐Analyzed Threshold Ionization (MATI) 21 D. Threshold Ion Separation by Kinetic Energy Differences 21 1. In a Reflecting Potential 22 2. In a Double‐Stage Acceleration Setup 22 3. In an Ion Trap 22 III. Application of Threshold Ionization Techniques 24 A. Ion Spectroscopy 24 1. Molecules 24 2. Clusters 25 3. Cluster Ion Dissociation Thresholds and Binding Energies 25 4. Excited Electronic States of Molecular Cations 29 B. Intermediate‐State Dynamics 29 C. Rydberg State Dynamics 31 1. Resolving the MATI Peak 31 2. Field‐Induced Rydberg‐Core Coupling 32 D. State‐Selected Ion‐Molecule Reactions 32 IV. Summary and Outlook 32 Acknowledgments 33 References 33 Multi‐photon excitation in a time‐of‐flight mass spectrometer (TOF‐MS) is shown to lead to threshold ions with defined internal energy. A powerful technique for the production of threshold ions is based on the excitation of high long‐lived Rydberg states embedded in the ionization continuum. The Rydberg molecules are separated with suitable separation techniques from ions produced by a direct multi‐photon ionization process. Finally, the ionization of the Rydberg molecules in a delayed pulsed electric field leads to threshold ions. This work reviews several separation techniques, and reports on applications of threshold ionization for investigation of the structure, energetics, and dynamics of neutral molecules, molecular cations, and cluster cations. © 2002 Wiley‐Liss, Inc. © 2002 Wiley Periodicals, Inc. Mass Spec Rev 21:16–36, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10014