Calcium (Ca) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca signals are decoded into defined plant adaptive responses by different Ca sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca-decoding and Ca-controlled target binding mechanisms in plants.