Background: FPs, including 5-fluorouracil and capecitabine, are widely used to treat solid tumor malignancies like gastrointestinal (GI) cancers. One-third of patients (pts) develop severe toxicities which may lead to treatment delays or hospitalization. Toxicities can be partly due to genetic variations in DPYD. Herein, we describe the implementation of an in-house DPYD test and its impact on FP dosing at a multisite cancer hospital. Methods: This is an observational study of pts starting or continuing FP-based chemotherapy who received DPYD testing as part of routine care. Buccal swabs were collected in clinic and sent to an in-house lab for genotyping using a polymerase chain reaction-based Drug Metabolizing Enzyme assay that interrogates all 5 variants with moderate-to-strong evidence according to the Clinical Pharmacogenetics Implementation Consortium (CPIC) (c.1905+1G>A, c.2846A>T, c.1679T>G, c.1236G>A and c.557A>G). Phenotype translations and dosing were based on CPIC guidelines. Test results and dose recommendations were uploaded to the electronic medical record (EMR) and emailed to the care team. In April 2022, pre- and post-test EMR alerts were built to prompt test ordering and dose modifications. The primary objective was to evaluate the proportion of DPYD variant carriers in tested pts receiving FP. Secondarily, we evaluated turnaround time and impact on FP dosing. Results: From March 2020-December 2022, 491 pts across 14 clinics received DPYD genotyping (54% male, 74% White, 20% Black, median age 53 years). GI cancers (~50% colorectal) represented 90% of the diagnoses. The median lab turnaround time was 3 (IQR 2-6) days. Pretreatment testing was ordered in 389 (79%) pts, of which 360 (93%) had results before cycle 1. Overall, 30 pts (6.1%) were heterozygous carriers (4.9% in pretreatment and 10.8% in reactive testing groups). Variants observed were c.1236G>A (n=13), c.2846A>T (n=8), c.1905+1G>A (*2A) (n=4), c.557A>G (n=4), and c.1679T>G (*13) (n=1). FP dose was modified in 27 (90%) pts (reduced in 25, avoided in 1, discontinued in 1). Of 19 carriers with pretreatment testing, 17 (90%) received an upfront dose reduction (mean reduction 42%; 3 had subsequent dose escalations); 1 avoided FP and 1 declined chemotherapy. Of 11 carriers with reactive testing, 9 had dose reductions (mean reduction 39%) and 2 discontinued FP due to toxicity. The mean FP dose intensity at cycle 1 was 56% in pretreatment carriers, 97% in reactive carriers, and 94% in wild type pts. Conclusions:DPYD genotype-guided FP dosing is feasible at a multisite cancer hospital. In-house rapid turnaround DPYD testing identified variant carriers and resulted in treatment/dose modifications for most carriers, potentially avoiding or mitigating severe toxicities and/or hospitalization, particularly with pretreatment testing. Toxicity evaluation is ongoing.