The excessive and uncontrolled release of neutrophil extracellular traps (NETs) is increasingly linked to the pathogenesis of various inflammatory diseases, cardiovascular disorders, and cancers. Real-time, non-invasive detection of NETs is crucial for understanding their role in disease progression and developing targeted therapies. Current NETs detection methods often lack the necessary specificity and resolution, particularly in vivo and ex vivo settings. To address this, we have developed novel near-infrared squaraine-peptide conjugates by rational molecular design as reporters of NETosis by targeting the protease activity of neutrophil elastase (NE). These self-quenching, cell-impermeable probes enable the precise real-time detection and imaging of NETs. The Förster resonance energy transfer (FRET)-based probe,