Background: Pre-analytical errors account for the majority of failures across the total testing process. This study evaluated pre-analytical performance in a tertiary-care biochemistry laboratory in Riyadh, Saudi Arabia, using IFCC-aligned Quality Indicators (QIs), Six Sigma metrics, and structured Root Cause Analysis (RCA). Methods: A retrospective analysis of all biochemistry tests processed between January and December 2024 at a tertiary-care hospital was conducted. Rejected tests were classified into seven IFCC-aligned QI categories. Sigma metrics assessed process capability, Pareto analysis identified the vital few contributors, and RCA using the Ishikawa framework identified human, equipment, environmental, and process-related factors. Rejection patterns were described by department and work shift. Results: Of 845,647 tests performed, 10,783 (1.28%) were rejected, yielding an overall process capability of 3.89σ (Minimum Acceptable). Hemolysis was the leading cause (8186 tests; 75.92%) at 3.97σ, the only indicator classified as High against the IFCC WG-LEPS registry. The remaining six indicators demonstrated Good to Very Good performance (4.59σ–5.33σ). Pareto analysis identified hemolysis and inappropriate tube use (7.60%) as the vital few, jointly responsible for 83.52% of rejections. RCA implicated venipuncture technique, needle gauge selection, workload pressure, and prolonged tourniquet application as key contributors. The Emergency Department generated the highest inpatient rejection burden (38.7%). Conclusions: Although the overall rejection rate compared favorably with international benchmarks, hemolysis was the principal process vulnerability, with inappropriate tube selection as a secondary target. Recommended quality improvement strategies include structured phlebotomy training, real-time hemolysis index feedback, and Emergency Department-specific initiatives; structural solutions such as dedicated inpatient phlebotomy services warrant prospective evaluation alongside training-based interventions.