Stigma involves discrediting a person or group based on a perceived attribute, behaviour or reputation associated with them. Sex workers (SW) and men who have sex with men (MSM) are key populations who are often at increased risk for the acquisition and transmission of HIV and who are affected by stigma that can negatively impact their health and well-being. Although stigma was included as an indicator in the US National HIV/AIDS Strategic Plan and there have been consultations focused on adding a stigma indicator within PEPFAR and the Global Fund in relation to potentiating HIV risks among key populations, there remains limited consensus on the appropriate measurement of SW- or MSM-associated stigma. Consequently, this systematic review summarizes studies using quantitative, qualitative, or mixed methods approaches to measure stigma affecting sex workers and men who have sex with men.This systematic review included English, French, and Spanish peer-reviewed research of any study design measuring SW- or MSM-associated stigma. Articles were published from January 1, 2004 to March 26, 2014 in PsycINFO, PubMed, EMBASE, CINAHL Plus, Global Health, and World Health Organization Global Health Library Regional Indexes. Of the 541 articles reviewed, the majority measured stigma toward MSM (over 97%), were conducted in North America, used quantitative methods, and focused on internalized stigma.With the inclusion of addressing stigma in several domestic and international HIV strategies, there is a need to ensure the use of validated metrics for stigma. The field to date has completed limited measurement of stigma affecting sex workers, and limited measurement of stigma affecting MSM outside of higher income settings. Moving forward requires a concerted effort integrating validated metrics of stigma into health-related surveys and programs for key populations.