With both compounds confirmed to be photoactive under cell culture conditions, we proceeded to investigate how they could be used in tandem to demonstrate a high degree of both spatial and temporal control of calcium signaling. To accomplish this we loaded both WinterGreen-WAY and WinterRed-NDMC at a concentration of 10 µM into cell culture media, chose a region of cells to activate by 488 nm uncaging, then immediately upon activation irradiated the same region with 639 nm light. The result of which is markedly tighter control of calcium flux durations and amplitude (Figure ). Using low doses of commercially available WAY-161503 spike into the cell culture media, we were not able to consistently reproduce such short calcium flux durations. Therefore, this represents not only a novel avenue to reproducibly control the activation and signaling pattern of the 5HT2C receptor but allows precise spatial control of the population induced. Photoactivatable forms of WAY-161503 and NDMC were designed as a system that allows for spectral multiplexing enabling tight spatiotemporal control of 5HT2C calcium signaling. WinterGreen-WAY and WinterRed-NDMC were synthesized, characterized, and tested for loss of bioactivity at physiologically relevant concentrations. The kinetics of photolysis were characterized using HPLC and confirmation of functional drug release was performed in live cell culture. Both compounds had acceptable photolysis half-lives of 2.792-7.472 seconds for WinterGreen-WAY and 12.99-20.73 seconds for WinterRed-NDMC, and quantitatively photolyzed in under 75 seconds. Importantly, both compounds remained stable in solution for 48 hours in the dark, and WinterGreen-WAY could release physiologically relevant payloads with a 200 ms pulse of 488 nm light in cell culture media. WinterGreen-WAY mediated the activation of 5HT2C signaling similar to that of commercially available WAY-161503, and with repeated 200 ms pulses of 488 nm light demonstrated longer calcium flux durations than that of 5 µM WAY-161053. This indicated light-dose dependent signaling for 5HT2C that could be achieved without deterioration of the response or high global drug concentration. Upon 639 nM irradiation, WinterRed-NDMC was demonstrated to diminish the effect of up to 5 µM WAY-161503 injected into cell culture media. Importantly, tandem uncaging of WinterGreen-WAY then WinterRed-NDMC produced markedly shorter calcium flux duration and amplitude, which was not reproducible with low dose commercially available WAY-161503. These tools enable a greater degree of spatiotemporal control of 5HT2C modulation and will allow for more detailed studies of the receptors signaling, interactions with other proteins, and native physiology. Future studies will focus on applying this system to spatiotemporally control synaptic transmission of primary neurons on a receptor subtype specific level.