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Background/Objectives: This study investigated whether serum salusin-α and salusin-β levels could support the diagnosis and prognosis of confirmed acute pulmonary embolism (APE) cases. Methods: A prospective observational study was conducted including 57 patients diagnosed with APE using computed tomography pulmonary angiography (CTPA) and 30 control participants without any acute or chronic disease. APE patients were categorized based on the Pulmonary Artery Obstruction Index (PAOI) into low (≤20) and high (>20) thrombus burden groups. Serum salusin-α and salusin-β levels were measured at diagnosis using an enzyme-linked immunosorbent assay. Associations with PAOI and Pulmonary Embolism Severity Index (PESI) scores were analyzed. Results: Salusin-α and salusin-β levels were markedly reduced in APE patients versus controls (p < 0.001). In multivariate analysis, salusin-α remained independently associated with APE (p = 0.042), whereas salusin-β was not significant. A receiver operating characteristic analysis showed good diagnostic performance for salusin-α (AUC = 0.799; sensitivity = 89.5%; specificity = 46.7%). Neither peptide correlated with PAOI or PESI. At a 305.85 pg/mL cut-off, salusin-α yielded a positive predictive value of 76.1% and a negative predictive value of 70% in this cohort. Conclusions: The findings suggest that salusin-α has high sensitivity in detecting acute pulmonary embolism and may serve as a supportive diagnostic marker in emergency settings. Although its specificity is limited, it could contribute to guiding additional testing. While salusin-β showed no significant diagnostic value, the potential role of salusin peptides in prognostic evaluation requires further exploration. Full article