Neuronal activity is accompanied by a net outflow of potassium ions (K⁺) from the intra- to the extracellular space. While extracellular [K⁺] changes during neuronal activity are well characterized, intracellular dynamics have been less well investigated due to lack of respective probes. In the current study we characterized the FRET-based K⁺ biosensor lc-LysM GEPII 1.0 for its capacity to measure intracellular [K⁺] changes in primary cultured neurons and in mouse cortical neurons in vivo. We found that lc-LysM GEPII 1.0 can resolve neuronal [K⁺] decreases in vitro during seizure-like and intense optogenetically evoked activity. [K⁺] changes during single action potentials could not be recorded. We confirmed these findings in vivo by expressing lc-LysM GEPII 1.0 in mouse cortical neurons and performing 2-photon fluorescence lifetime imaging. We observed an increase in the fluorescence lifetime of lc-LysM GEPII 1.0 during periinfarct depolarizations, which indicates a decrease in intracellular neuronal [K⁺]. Our findings suggest that lc-LysM GEPII 1.0 can be used to measure large changes in [K⁺] in neurons in vitro and in vivo but requires optimization to resolve smaller changes as observed during single action potentials.