Abstract: A fundamental challenge in calcium imaging is to minimize the excitation laser power while still maintaining a sufficient signal-to-noise ratio to distinguish individual transients in the fluorescence traces. It is important to characterize relative fluorescence (i.e., ΔF/F) dependence on the excitation wavelength in vivo where the environment cannot be controlled effectively during imaging. Leveraging time division multiplexing of two excitation beams to achieve nearly simultaneous 2-photon and 3-photon imaging of the calcium transients, we measured systematically the ΔF/F dependence on the excitation wavelength in 2-photon and 3-photon in vivo imaging of neuronal activity in mouse brain labeled with GCaMP6s. The technique can be applied to in vivo measurements of other fluorescence sensors.