Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.