Over the past 10 years, the Peruvian National Tuberculosis (TB) Program, the National Reference Laboratory (NRL), Socios en Salud, and US partners have worked to strengthen the national TB laboratory network to support treatment of multidrug-resistant TB. We review key lessons of this experience. The preparation phase involved establishing criteria for drug susceptibility testing (DST), selecting appropriate DST methods, projecting the quantity of DST and culture to ensure adequate supplies, creating biosafe laboratory facilities for DST, training laboratory personnel on methods, and validating DST methods at the NRL. Implementation involved training providers on DST indications, validating conventional and rapid first-line DST methods at district laboratories, and eliminating additional delays in specimen transport and result reporting. Monitoring included ongoing quality control and quality assurance procedures. Hurdles included logistics, coordinating with policy, competing interests, changing personnel, communications, and evaluation. Operational research guided laboratory scale-up and identified barriers to effective capacity building.