Introduction: Measuring performance indicators is a critical element of Quality Assurance for any diagnostic test as well as an ISO 15189 requirement. Targets should be set for all indicators monitored, and any unexplained change in quality indicators, such as an increase in error rates, a change in MTB positivity rate or Rifampicin resistance rate, or a significant change in volume of tests conducted, should be documented and investigated. Objective: The aim of this study was to establish and implement Quality indicators for Mycobacteriology Laboratory at PHIL, Hawassa, South Ethiopia from October 27, 2020 to February30, 2021. Method: A cross-sectional study was conducted on 229 samples from presumptive TB cases for Gene Xpert MTB/Assays and 439 specimens for MGIT and LJ Culture, to assess the Quality Indicators status of each technique. The specimens were received from Facilities under the referral network to the Mycobacteriology and processed, examined by the Mycobacteriology Lab as per the standard operating procedure and Data were tracked using real-time Gene Xpert software, Laboratory registers, and standardized formats. Fulfillment for Quality indicators was evaluated. Results: Of the 239 sputum sample processed by the Gene Xpert MTB/Assay, the M.Tuberculosis detection rate among presumptive TB cases was 37/229(16.2%), Rifampicin resistant tuberculosis was 1(2.7%) and the overall unsuccessful rate was 4/229(1.7%). The mean TAT for Gene Xpert MTB/RIF assay result is 21hrs with SD±6hours. While ,439 sputum specimens processed using both LJ and BACTEC MGIT culture methods the recovery rate for smear positive samples for both LJ and BACTEC MGIT Culture methods was 19/35(54.3%), whereas the recovery rate for smear negative samples was 19/369(5.1%) and 16/(4.2%) for BACTEC MGIT 960 and LJ methods respectively. Specimen rejection rate calculated was 0.93% (4/439).The mean TAT of mycobacteria from smear positive samples was 14.8 and 35.6, with SD±7.25 and 9.55 days for BACTEC MGIT 960 and LJ methods respectively. TAT for smear negative samples was 18.9 and 38.7 days for BACTEC MGIT 960 and LJ methods, with SD±3 and 5days respectively. The Contamination rates were 45/439(10.2%) and 42/439(9.6%) for liquid and solid cultures respectively. Non-Tuberculosis Mycobacteria detection rate was 6/439 (1.4%) Conclusion: Majority of the quality indicators are within target for specimen rejection, turnaround times for result delivery, Non-tuberculosis mycobacteria detection rates, Xpert MTB detection, and unsuccessful rates, whereas demands due attention to improve those indicators which were out of the target, low recovery rate, culture contamination rates and turnaround time to smear negative culture results.