Background In 2011, World Health Organization revised its recommendation for microbiological monitoring during treatment for multidrug-resistant tuberculosis (MDR-TB) by increasing the frequency of culture examination from quarterly to monthly after culture conversion. Implementing the recommendation requires substantial additional investment in laboratory infrastructure. The objective of this review is to provide cost evidence that is needed for national TB programs to budget for optimal monitoring strategies. Methods and Findings We conducted the first systematic literature review on unit cost estimates of three monitoring strategies: 1) smear only; 2) culture only; 3) combined smear and culture. 26 peer-reviewed studies were selected by searching 10 databases in English and Chinese for literature published between 1995 and 2012. Cost estimates were converted into 2010 constant USD and international dollars. We assessed the quality of the estimates using a matrix with five essential elements and provided a cost projection for the combined smear and culture tests where the data were available. The 26 studies reported the cost estimates in 16 predominantly high-or middle-income countries from 1993 to 2009. The estimated unit cost for smear, culture, and combined tests ranges from 0.26to 10.50, 1.63to 62.01, and 26.73to 39.57, respectively. The ratio of culture to smear costs varies from 1.35 to 11.98. The wide range of estimates is likely attributable to using different laboratory methods in different regions and years and differing practices in collecting and reporting cost data. Most studies did not report information critical for generalizing their conclusions. Conclusion The paucity and low quality of unit cost estimates for TB monitoring in resource-poor settings impose technical challenges in predicting the resources needed for strengthening microbiological monitoring. To improve the validity and comparability of the cost data, we strongly advocate the data collection, estimation, and reporting follow protocols proposed by WHO.