Fiber photometry is a key technique for characterizing brain-behavior relationships in vivo. Initially, it was primarily used to report calcium dynamics as a proxy for neural activity via genetically encoded indicators. This generated new insights into brain functions including movement, memory, and motivation at the level of defined circuits and cell types. Recently, the opportunity for discovery with fiber photometry has exploded with the development of an extensive range of fluorescent sensors for biomolecules including neuromodulators and peptides that were previously inaccessible in vivo. This critical advance, combined with the new availability of affordable "plug-and-play" recording systems, has made monitoring molecules with high spatiotemporal precision during behavior highly accessible. However, while opening exciting new avenues for research, the rapid expansion in fiber photometry applications has occurred without coordination or consensus on best practices. Here, we provide a comprehensive guide to help end-users execute, analyze, and suitably interpret fiber photometry studies.