Lung cancer is the leading cause of cancer-related deaths and the second most prevalent cancer type in the United States. Most lung cancers are subcategorized as non-small cell lung cancers (NSCLC) which are primarily treated by surgical resection. The nonspecific nature of identifying NSCLCs intraoperatively results in a high rate of disease recurrence in these patients. In this thesis, we translated the phospholipase A2 (PLA2) activatable fluorophore, DDAO-arachidonate (DDAO-A), from triple-negative breast cancer (TNBC) to NSCLC imaging due to the high mortality rate associated with lung cancer and the overactivity of cytosolic PLA2 (cPLA2) reported in NSCLCs. We hypothesized that DDAO-A would be preferentially activated in lung cancer models and tissues compared to normal and background tissues. To test this hypothesis, we first optimized the liposomal formulation determining that 100 nm membrane-extruded egg phosphatidylcholine (EggPC) liposomes exhibited the best physical properties with a low average size (105.7 nm) and polydispersity index (PDI; 0.094) and a zeta potential within the designated -5 to -15 mV range. Further, the EggPC liposomes exhibited the highest maximum fluorescence and most rapid activation of DDAO-A of the formulations tested. Stability of the formulation when stored as a lipid film was investigated demonstrating optical stability over four weeks. DDAO-A activation was then tested in vitro and in vivousing recombinant human cPLA2, NSCLC cells, and NSCLC tumor-bearing mice. Fluorescence was consistently higher when in the presence of recombinant cPLA2 and NSCLC cells versus nonspecific hydrolysis. Additionally, comparison with the activation of the secretory PLA2 (sPLA2) specific fluorophore, DDAO-palmitate (DDAO-P), demonstrated preferential activation in tumor-bearing mice treated with DDAO-A compared to the control suggesting activation in NSCLC is likely mediated by cPLA2 activity. DDAO-A activation was evaluated ex vivo using human and canine lung tumor and normal lung tissues demonstrating a preferential activation of the fluorophore in 8 out of 10 human tumors and all 3 canine tumors as evidenced by tumor-to-normal ratios (TNR) of 2:1 or higher. The preferential and rapid activation of DDAO-A in NSCLC models demonstrated a novel method for NSCLC imaging in real-time following topical administration for tumor margin delineation.