Current tools to annotate protein function have failed to keep pace with the speed of DNA sequencing and exponentially growing number of proteins of unknown function (PUFs). A major contributing factor to this mismatch is the historical lack of high-throughput methods to experimentally determine biochemical activity. Activity-based methods, such as activity-based metabolite and protein profiling, are emerging as new approaches for unbiased, global, biochemical annotation of protein function. In this review, we highlight recent experimental, activity-based approaches that offer new opportunities to determine protein function in a biologically agnostic and systems-level manner.