Background: The accuracy of clinical laboratory results is paramount for effective patient diagnosis and treatment. However, errors in the pre-analytical phase, particularly during phlebotomy, significantly impact the reliability of laboratory data. With the Clinical and Laboratory Standards Institute (CLSI) guidelines serving as a benchmark for best practices in blood collection, adherence to these standards is crucial for minimizing errors. Objective: This study aimed to assess compliance with modified CLSI guidelines in phlebotomy procedures within a healthcare setting and identify the most prevalent errors in the pre-analytical phase that could compromise patient safety and the integrity of laboratory results. Methods: Conducted at the Combined Military Hospital Lahore from January to March 2023, this observational study scrutinized phlebotomy practices across three distinct healthcare settings: emergency department, outpatient department, and clinical wards. A structured checklist, adapted from the European Federation for Clinical Chemistry and Laboratory Medicine (EFLM) guidelines and aligned with local modifications, was employed to evaluate compliance in 20 specific areas of phlebotomy practice. A total of 285 phlebotomies were observed by specialized staff, with data analyzed using SPSS version 25 to compare error frequencies and compliance rates across different settings and among various healthcare professionals. Results: The study revealed an overall compliance rate of 88% for identified request forms, 76% for patient identification according to local guidelines, and 66% for proper hand sanitization. However, significant errors were identified in checking for potential complications of venipuncture (34% compliance), with tubes being clearly under or overfilled (46% compliance), and in verifying that patients were prepared for phlebotomy (46% compliance). The error frequency varied across settings, with the highest discrepancies observed in the emergency department. Differences in compliance rates between laboratory staff and nurses were statistically significant in key areas, including patient identification (p<0.001) and tube labeling (p=0.081). Conclusion: The study highlights a critical need for improving adherence to CLSI guidelines within phlebotomy practices to enhance patient safety and the accuracy of laboratory results. Targeted educational and training interventions are essential for addressing the identified gaps in compliance, particularly in patient identification, sample volume control, and verification of patient preparation for phlebotomy.