Necrotizing enterocolitis (NEC) remains a leading cause of premature newborn morbidity and mortality. In order to make progress in preventing NEC through clinical studies and trials, significant challenges in disease risk prediction and diagnostic ambiguity need to be addressed. Clinical risk association and diagnostic models are emerging, along with promising advances in biomarker studies. New molecular markers, diagnostic imaging modalities, and novel platforms to interrogate gut function may qualify clinical biomarkers, as well as increase the basic understanding of NEC pathophysiology. Broader validation studies are needed in order to determine the clinical utility of several promising biomarker targets. Biochemical indices residing at the intersection of metabolism and enteric microbiome studies are among the most recent and promising areas of investigation driving clinical understanding. Taken together, the combination of new modality metrics, along with robust clinical association models, holds promise for addressing the unmet needs of improved NEC risk prediction and diagnosis.