Ca2+ is a ubiquitous secondary messenger involved in numerous plant signaling transduction cascades, such as defense responses to environmental cues including abiotic and biotic stresses, gravitropism, pollen tube growth, nodulation, etc. Ligand binding to a cognate receptor triggers a cytosolic Ca2+ spike required for induction of downstream signaling. The two major ion channel families that have been identified in plants to conduct Ca2+ are cyclic nucleotide gated channels (CNGC) and ionic glutamate receptors (GLR). Members in both families have been characterized to conduct Ca2+ into the cytosol. A substantial aspect of CNGC signaling in plants involves CNGC-dependent cytosolic Ca2+ spikes that then are transduced into both long-term but also (often underappreciated) instantaneous signal responses. CNGCs are activated by cyclic nucleotides, cAMP and cGMP. Calmodulin and calmodulin-like proteins regulate CNGC channel function by binding to the calmodulin binding domains in CNGC. Cyclic nucleotides are also universal secondary messengers. Although evidence has suggested the existence of cyclic nucleotide signaling in plant cells, none of the enzymes responsible for their biosynthesis and degradation have been identified. Optogenetic approaches for the determination of Ca2+ elevation in response to stimuli and techniques of studying cyclic nucleotide, CNGC, and GLR associated signaling in plant cells have been developed and advanced in the past two decades. This collection of such cutting-edge techniques will provide practical, instructive, and experiential tools for researchers in this field. It will also encourage communication and collaboration between scientists studying Ca2+ signaling in plants.