Background: Pancreatic cancer has an aggressive disease course, mandating close clinical monitoring while on treatment. Evaluation of treatment response in patients with mPDAC necessitates scheduled clinical and radiographic assessments along with monitoring serum CA 19-9 levels. We investigated the impact of serum CA 19-9 monitoring and its association with clinical outcomes in patients with mPDAC who received second- (2L) and third line (3L) in a population-based setting. Methods: Data were extracted from the nationwide Flatiron Health electronic health record (EHR)-derived de-identified database for patients diagnosed with mPDAC and subsequently treated in the 2L or 3L setting between January 1, 2014 and June 30, 2020. Serum CA 19-9 levels at baseline were extracted – defined as the values obtained ≤ 60 days of treatment initiation and during treatment. CA 19-9 levels > 40 IU/mL were considered elevated. Survival analysis was performed using Kaplan-Meier methods. Categorical measures were compared with a chi-square test and survival outcomes with a log-rank test. Results: There were 2,402 patients who received 2L and 790 patients who received 3L treatment included in the study. Among the 2L cohort, median age at treatment initiation was 67 years (IQR: 60 – 73), 54% were male, 57% had an ECOG score of 0-1, and 82% had a baseline serum CA 19-9 level available. Among the 3L cohort, median age at treatment initiation was 67 years (IQR: 60 – 73), 53% were male, 58% had an ECOG score of 0-1, and 84% had a baseline CA 19-9 level available. Most patients in the 2L and 3L cohorts had an elevated CA 19-9 at baseline, 85.2% and 82.0%, respectively. Among the 2L and 3L cohorts, 38.5% and 31.9% had CA 19-9 levels decrease/remain stable during treatment and 27.6% and 31.4% had levels increase during treatment, respectively. Patients with normal baseline CA 19-9 experienced longer survival than patients with elevated levels [2L: 7.2 months (95% CI: 6.1 – 9.2) vs 5.2 months (4.9 – 5.6), p < 0.001; 3L: 6.1 months (5.4 – 9.1) vs 3.9 months (3.4 – 4.3), p < 0.001]. Similarly, patients with decreasing/stable CA 19-9 during treatment had longer survival than patients who had their CA 19-9 levels increase [2L: 8.2 months (7.7 – 8.5) vs 4.3 months (4.1 – 4.7), p < 0.001; 3L: 7.5 months (6.6 – 9.2) vs 3.7 months (3.4 – 4.3), p < 0.001]. Conclusions: In this large, contemporary, real-world study of patients with mPDAC, CA 19-9 levels at treatment initiation had a prognostic value across later lines of therapy. Routine serial monitoring of CA 19-9 levels during treatment, in addition to clinical and radiographic assessment, may help with timely additional diagnostic testing and treatment intervention. Further validation studies are needed to understand the generalizability of these results.