The activity of neurons throughout the brain is determined by the precise relationship between excitatory and inhibitory inputs and disruption of this relationship underlies many psychiatric diseases. Whether these inputs covary over time or between repeated stimuli remains unclear due to the lack of experimental methods for measuring both inputs simultaneously. We developed a new analytical framework for simultaneous measurement of both the excitatory and inhibitory neuronal inputs during a single trial of a current clamp recording. This can be achieved by injecting a current composed of two high frequency sinusoidal components followed by analytical extraction of the conductances. We demonstrate the ability of this method to measure both inputs in a single trial under realistic recording constraints and from morphologically realistic CA1 pyramidal model cells. We next applied the method to in-vitro electrophysiological recordings, using optogenetics to evoke mixed inhibitory and excitatory synaptic responses, and achieved high resolution extraction of both excitatory and inhibitory conductances in a single trial. Importantly, these conductances were similar both in amplitude and time course to the conductances that we measured from the same cells using classical voltage clamp recordings - fully validating our new method. We present this method as a new experimental tool, which will facilitate our understanding of fundamental questions in neuroscience, in both health and disease.