With an estimated 9.4 million new cases globally, tuberculosis (TB) continues to be a major public health concern. Eighty percent of all cases worldwide occur in 22 high-burden, mainly resource-poor settings. This devastating impact of tuberculosis on vulnerable populations is also driven by its deadly synergy with HIV. Therefore, building capacity and enhancing universal access to rapid and accurate laboratory diagnostics are necessary to control TB and HIV-TB coinfections in resource-limited countries. The present review describes several new and established methods as well as the issues and challenges associated with implementing quality tuberculosis laboratory services in such countries. Recently, the WHO has endorsed some of these novel methods, and they have been made available at discounted prices for procurement by the public health sector of high-burden countries. In addition, international and national laboratory partners and donors are currently evaluating other new diagnostics that will allow further and more rapid testing in point-of-care settings. While some techniques are simple, others have complex requirements, and therefore, it is important to carefully determine how to link these new tests and incorporate them within a country's national diagnostic algorithm. Finally, the successful implementation of these methods is dependent on key partnerships in the international laboratory community and ensuring that adequate quality assurance programs are inherent in each country's laboratory network.