Grimace scales have been used for pain assessment in different species. This study aimed to develop and validate the Feline Grimace Scale (FGS) to detect naturally-occurring acute pain. Thirty-five client-owned and twenty control cats were video-recorded undisturbed in their cages in a prospective, case-control study. Painful cats received analgesic treatment and videos were repeated one hour later. Five action units (AU) were identified: ear position, orbital tightening, muzzle tension, whiskers change and head position. Four observers independently scored (0-2 for each AU) 110 images of control and painful cats. The FGS scores were higher in painful than in control cats; a very strong correlation with another validated instrument for pain assessment in cats was observed (rho = 0.86, p < 0.001) as well as good overall inter-rater reliability [ICC = 0.89 (95% CI: 0.85-0.92)], excellent intra-rater reliability (ICC > 0.91), and excellent internal consistency (Cronbach's alpha = 0.89). The FGS detected response to analgesic treatment (scores after analgesia were lower than before) and a cut-off score was determined (total pain score > 0.39 out of 1.0). The FGS is a valid and reliable tool for acute pain assessment in cats.