Understanding the dynamic changes in the intracellular metabolism of immune cells has become fundamental to understanding the regulation of their effector functions. Optical metabolic imaging, consisting of optical redox ratio and fluorescence lifetime imaging microscopy of endogenous coenzymes NAD(P)H and FAD, offers a label-free and non-invasive approach to assess intracellular metabolism at the single-cell level. The major advantage of optical metabolic imaging is that it can assess heterogeneity in the sample with spatiotemporal resolution. While this approach has been mainly used to perform metabolic imaging on in vitro samples, studies have demonstrated that it also performs well in live, intact animals, and is sensitive to dynamic changes in immune cell activation. This chapter describes protocols for performing optical metabolic imaging of innate immune cells at the caudal fin wound microenvironment of larval zebrafish following sterile injuries. However, the protocol can be readily applied to other cell types and in different biological contexts.