Purpose <p>The study aims to critically review US FDA warning letters (WLs) issued during inspections between 2019 and 2024 for compliance with Good Laboratory Practice in Preclinical research facilities.</p> Methods <p>WLs were downloaded from the US FDA public domain of the WLs and the content of each WL was assessed based on types and frequencies of violations and corrective and preventive actions (CAPA) employed by the testing facility or requested by the FDA investigators. Each WL was evaluated and critically analyzed about the applicable regulation. Violations were correlated with consequences of non-compliance, as addressed by the FDA inspectors.</p> Results <p>Eight WLs letters were analyzed. The incidence of violations concerning the QAU’s failure to meet responsibilities, documentation deficiencies, noncompliance with study protocols, deviations in animal handling, and data capture in the final study report was 12% each. The violations concerning the study director’s failure to fulfil his/her duties and deficient/ noncompliance to pertinent SOPs were 10% each. The incidence of inadequate qualifications among study personnel and the management of specimens/reagents and controls was 9% and 6%, respectively. The minimal variance was linked to specimen/record retention (5%).</p> Conclusion <p>Testing facilities in preclinical research are requested to evaluate personnel responsibilities, improve GLP training programs, and implement adequate standard procedures to comply with GLP standards, reducing the recurrence of these violations and enhancing data integrity and validity that will support a clinical submission.</p>