Biomarkers have the potential to significantly change diagnostic strategies and influence therapeutic management. We developed a MALDI-TOF protein expression profiling platform for biomarker discovery and a proof-of-principle study identified two proteins, cyclophilin A (CyPA) and macrophage migration inhibitory factor (MIF), that were overexpressed in non-small cell lung cancer (NSCLC). The current study focused on evaluating the potential of CyPA and MIF as prognostic markers in patients with a new diagnosis of lung cancer for rapid translation into clinical practice. Two hundred and thirty-four primary NSCLC specimens reflecting a broad range of histologies and stages were examined for CyPA and MIF reactivity by tissue microarray immunohistochemistry (TMA-IHC). The percent tumor cell reactivity, staining intensity and a composite staining score were compared with overall patient survival by Kaplan-Meier curves, log rank test and Cox model statistics. Although both proteins were overexpressed in most NSCLC tumors, neither CypA nor MIF showed a correlation with outcome. This pilot project approach can expedite integration of newly discovered biomarkers into clinical practice, with the goal of improving stratification of patients into appropriate treatment regimens. While both proteins considered in this study were overexpressed in the vast majority of NSCLCs, they were not found to be of prognostic significance.