Through the highly regulated events of intracellular Ca²⁺ homeostasis, often seen as a Ca²⁺, wave or spike, intracellular Ca²⁺ acts as a second messenger that regulates numerous physiological cellular phenomena including fertilization, development and apoptosis. To study these Ca²⁺-mediated cellular processes, multidisciplinary approaches were employed combining information ranging from sequence to structure to cellular dynamics. First, the impressive growth of sequence databases inspired a bioinformatics approach to studying the function of large Ca²⁺-mediated protein superfamilies. To improve function prediction, a bioinformatics algorithm was developed for classifying subfamilies of large protein superfamilies and was demonstrated on the EF-hand superfamily. Using this algorithm combined with an understanding of the domain architecture of subfamilies, a classification tool and web-accessible database was created for the cadherin superfamily of Ca²⁺-dependent adhesion molecules (http://calcium.uhnres.utoronto.ca/cadherin). Though rapid, these sequence-based approaches can only vaguely predict Ca²⁺-mediated functions, while a higher resolution understanding would require spatio-temporal imaging of Ca²⁺, signals in living cells. Thus, the second part of this thesis is focused on imaging Ca²⁺-related signaling events using FP (fluorescent protein)-based ratiometric FRET (fluorescence resonance energy transfer) probes (hereafter, FP-based probes). Using the atomic structure of the CaM (calmodulin) in complex with CKKp (CaM kinase kinase peptide), a new FP-based probe was modeled and created with improved characteristics for imaging of Ca 2+ signaling. In in vivo imaging, this new design resulted in a two-fold enhancement in the FRET efficiency, thereby expanding the ability of Ca²⁺ FP-based probe to visualize Ca²⁺ mobilization in living cells where a higher sensitivity is required for detection. Similarly, probes were created for detecting caspase proteolytic cleavage and using a co-culture of cells expressing different FP-based probes, simultaneous Ca²⁺ signaling and caspase cleavage events were imaged in cell death induced by an oxidative burst modeled by a bolus addition of [H₂O₂]_f = 10 mM. When COS-7 cells were induced by an oxidative burst, caspase activation was observed within 2 minutes. This is faster than previously thought and could not been observed using standard in vitro caspase cleavage kits.