In the last 20 years there have been remarkable advances in our ability to track movement and activities of proteins within cells. This is largely due to improved chemical probes and fluorescent proteins, and technical advances in microscopy. A remaining challenge is real-time multiplexed imaging. Excitation scan-based hyperspectral imaging (HSI) approaches are well suited for multiplexed imaging. However, excitation scan-based HSI has not been widely adopted, in part due to a lack of protocols for selection of combinations of fluorescent labels and proteins, and determining the range of excitation wavelengths and dichroic filters. Here we address this issue by outlining considerations for the selection of multiple labels for excitation scan-based HSI. HEK-293 cells were transfected with fluorescent protein constructs and/or loaded with dyes or labels for measurement of excitation spectra. Cells were imaged using a custom-built excitation scan-based HSI microscope that utilizes tunable thin film filters to filter fluorescence excitation from 360 nm to 550 nm in 5 nm increments in conjunction with a long pass dichroic filter and long pass emission filter. We observed that we can effectively quantify the relative abundance and spatial distributions of NucBlue, AlexaFluor 488, AlexaFluor 514, and AlexaFluor 555, Cal520, Cal590, as well as the fluorescent proteins GFP, Cerulean, Turquoise, Venus, tdTomato, and mCherry, individually and in combinations. We are currently assessing the spectra of these fluorophores using excitation scan-based HSI microscope systems.