Abstract Background Quality assurance is vital in laboratory diagnostics to ensure patient safety and accurate results. Laboratories employ internal and external quality control measures, particularly focusing on the analytical phase. Sigma metrics, representing process variation, play a crucial role in assessing accuracy, with Six Sigma setting high standards for operational excellence. This study aims to enhance laboratory testing procedures by analyzing the analytical phase. In 2022, the Emergency laboratory at KFAFH, Jeddah, achieved optimal sigma performance, marking a significant improvement in analytical testing. http://www.westgard.com/sigma-vp-kfafh.htm. In 2023, The study also demonstrated a 30% productivity increase, driven by sustained efforts and Sigma metrics implementation, leading to cost reductions and improved quality. Real-time patient moving averages enhance quality assurance, extending coverage to the total testing process. The primary objectives include redesigning IQC procedures and optimizing resource utilization. This will be accomplished by reducing the frequency of controls, minimizing unnecessary reruns, optimizing resource allocation, and managing total costs, all while ensuring compliance and averting violations. Methods A retrospective-prospective study was conducted at the Emergency Laboratory of King Fahad Armed Forces Hospital in Jeddah, Saudi Arabia, from May 2021 to October 2023. The study assessed 841,558 tests for 131,785 emergency patients. Forty-seven analytes were evaluated for Internal Quality Control and External Quality Control (CAP), with monthly data collection. Chemistry and Hematology parameters were analyzed using Unity Real-Time software on Alinity-ci and Alinity-hq analyzers. Data was collected in three stages: baseline, pre-Sigma verification, post-Sigma implementation, and a sustainability phase. The methodology involved DMAIC model tools like flowcharts, cause & effect analysis, and sigma metric scales. Six Sigma process tools, including DPM and process capability analysis, were used. Interventions included Six Sigma metrics for QC performance, DPM for TAT process improvement, and patient moving average Real-time for proactive risk management in reducing analytical testing variation. Results Excellent performance (> 6 sigma) was achieved for 70% of chemistry test parameters for three control levels.: This indicates a high level of process capability and reliability in laboratory operations, ensuring accuracy and consistency in test results. The study successfully reduced process variation in Emergency Room (ER) laboratory Turnaround times (TATs), leading to an increase in process capability as measured on the Sigma scale, which improved from 1.35 to 3.1, indicating a more robust and efficient laboratory operation. Reducing the cost and efficiency of internal failure controls by 29% and at the same time saving in labor cost/workload marked a decrease in QC frequency from 7.0% to 0.18%, reaching the target goal of TATs and satisfaction. Conclusions The adoption of Six Sigma methodologies resulted in a notable decrease in QC runs, signaling cost savings. These high-quality methods streamline laboratory operations, improve cost-effectiveness for the healthcare system, and ultimately benefit patients and clinicians. Six Sigma methodologies play a crucial role in selecting appropriate QC designs for each parameter, ensuring efficient control while maximizing cost-effectiveness. Emphasizing the importance of suitable QC strategies and continuous technologist training is vital for mitigating analytical errors and enhancing turnaround time through comprehensive automation of the analytical phase.