Significance The control of intracellular Cl ⁻ and pH plays a crucial role in several neuronal functions, and the study of these processes would be helped by tools for their noninvasive optical measurement in vivo. In this study, we have performed combined measurements of Cl ⁻ and pH of individual pyramidal neurons by means of in vivo two-photon imaging, and we provide direct experimental demonstration for the presence of the postnatal developmental shift to lower intraneuronal Cl ⁻ . Moreover, we introduce an approach for dynamic and simultaneous monitoring of intraneuronal Cl ⁻ and pH in vivo. These methods will open a window for the study of the roles of intraneuronal pH and Cl ⁻ in neuronal signaling, plasticity, and disease.