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Personalized approaches for continuous monitoring of chloride levels are potentially valuable for evaluating the efficacy of new treatments of genetic disorders such as cystic fibrosis. In this report, we validated optode-based nanosensors for real-time chloride monitoring in the interstitial fluid of living animals. These nanosensors take advantage of a ratiometric sensing scheme which demonstrates reversible and selective chloride detection in the physiological range. We further investigate how skin pigmentation affects the sensor performance during in vivofluorescence imaging. We successfully monitored endogenous chloride changes using nanosensors during pharmacological treatment in a cystic fibrosis mouse model. We believe this platform is a valuable tool for chloride detection which could assess the efficacy of new treatments for cystic fibrosis.