Intraneuronal chloride concentrations ([Cl-]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing GABA responses via chloride-permeable GABAA receptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl-]i using two-photon microscopy in brain slices. Depending on the binding of chloride, with this sensor, Fluorescence Resonance Energy Transfer (FRET) occurs from the CFP donor to the YFP acceptor. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We observed a clear reduction in [Cl-]i between day in vitro (DIV)3 and DIV22. To study the developmental decrease in [Cl-]i in inhibitory neurons, we used viral expression of the SClm sensor in VGAT-Cre mice. We observed that the developmental [Cl-]i reduction in interneurons was ~1 week delayed compared to pyramidal cells. In addition, we used the SClm sensor to assess alterations in [Cl-]i in the medial prefrontal cortex (mPFC) of postnatal day (P)9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift toward higher (i.e., immature) chloride levels in layer 2/3 pyramidal cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl-]i in slices.