|Background: Aspiration of stomach content or saliva in critical conditions—e.g., shock, intoxication, or resuscitation—can lead to acute lung injury. While various biomarkers in bronchoalveolar lavage fluids have been studied for diagnosing aspiration, none have been conclusively established as early indicators of lung damage. This study aims to evaluate the diagnostic value of pepsin, bile acid, and other biomarkers for detecting aspiration in an intensive care unit (ICU). Materials and methods: In this study, 50 ICU patients were enrolled and underwent intubation before admission. The evaluation of aspiration was based on clinical suspicion or documented instances of observed events. Tracheal secretion (TS) samples were collected within 6 h after intubation using sterile suction catheters. Additional parameters, including IL-6, pepsin, and bile acid, were determined for analysis. Pepsin levels were measured with an ELISA kit, while bile acid, uric acid, glucose, IL-6, and pH value in the tracheal secretion were analyzed using standardized lab methods. Results: The 50 patients admitted to the ICU with various diagnoses. The median survival time for the entire cohort was 52 days, and there was no significant difference in survival between patients with aspiration pneumonia (AP) and those with other diagnoses (p = 0.69). Among the AP group, the average survival time was 50.51 days (±8.1 SD; 95% CI 34.63–66.39), while patients with other diagnoses had a mean survival time of 32.86 days (±5.1 SD; 95% CI 22.9–42.81); the survival group comparison did not yield statistically significant results. The presence of pepsin or bile acid in TS patients did not significantly impact survival or the diagnosis of aspiration. The p-values for the correlations between pepsin and bile acid with the aspiration diagnosis were p = 0.53 and p > 0.99, respectively; thus, pepsin and bile acid measurements did not significantly affect survival outcomes or enhance the accuracy of diagnosing aspiration pneumonia. Conclusions: The early and accurate diagnosis of aspiration is crucial for optimal patient care. However, based on this study, pepsin concentration alone may not reliably indicate aspiration, and bile acid levels also show limited association with the diagnosis. Further validation studies are needed to assess the clinical usefulness and reliability of gastric biomarkers in diagnosing aspiration-related conditions. Such future studies would provide valuable insights for improving aspiration diagnosis and enhancing patient care.