Herein, we report the synthesis of a chemical probe for real-time visualization of L-type voltage-gated calcium channels (LTCCs) using fluorescence microscopy. The probe, FluoBar2, is based on a barbiturate ligand selective for LTCCs. FluoBar2 contains the organic fluorophore 5-carboxyfluorescein and was synthesized in seven total steps with a 22% overall yield.