Molecular imaging often relies on the use of targeted and activatable reporters to quantitate and visualize targets, biological processes, and cells in vivo. The use of optical probes with near-infrared fluorescence allows for improved photon penetration through tissue and minimizes the effects of tissue autofluorescence. There are several parameters that define the effectiveness of imaging agents in vivo. These factors include probe targeting, activation, pharmacokinetics, biocompatibility, and photophysics. Recent advances in our understanding of these variables as they pertain to the application of optical reporters for in vivo imaging are discussed in this review.