Whole-genome genetic screening, enabled by CRISPR-Cas systems, serves as a powerful tool for the identification of host genetic factors mediating biological processes. We have developed a platform for high-throughput whole-genome CRISPR screening of intracellular calcium signalling via FACS, allowing us to identify novel machinery used to transduce painful stimuli from virtually any chemical or biological agent. Here, we report the development of this assay and its application in a proof-of-concept genetic screen to identify novel mediators of capsaicin responses. This screen nominated three transmembrane proteins which are associated with various pain states in both humans and rodents and whose function is currently undergoing characterisation. Our platform can be readily adapted to investigate effectively any process involving calcium signalling events and is currently being used in elucidating the mechanism of action of a range of noxious agonists, including various venoms.