Background: Internet-based testing for sexually transmitted infections (STIs) allows asymptomatic individuals to order a self-sampling kit online and receive their results electronically, reducing the need to attend a clinic unless for treatment. This approach has become increasingly common; however, there is evidence that barriers exist to accessing it, particularly among some high-risk populations. We review the qualitative evidence on this topic, as qualitative research is well-placed to identify the complex influences that relate to accessing testing. Objective: This paper aims to explore perceptions and experiences of internet-based testing for STIs among users and potential users. Methods: Searches were run through 5 electronic databases (CINAHL, EMBASE, MEDLINE, PsycINFO, and Web of Science) to identify peer-reviewed studies published between 2005 and 2018. Search terms were drawn from 4 categories: STIs, testing or screening, digital health, and qualitative methods. Included studies were conducted in high-income countries and explored patient perceptions or experiences of internet-based testing, and data underwent thematic synthesis. Results: A total of 11 studies from the 1735 studies identified in the initial search were included in the review. The synthesis identified that internet-based testing is viewed widely as being acceptable and is preferred over clinic testing by many individuals due to perceived convenience and anonymity. However, a number of studies identified concerns relating to test accuracy and lack of communication with practitioners, particularly when receiving results. There was a lack of consensus on preferred media for results delivery, although convenience and confidentiality were again strong influencing factors. The majority of included studies were limited by the fact that they researched hypothetical services. Conclusions: Internet-based testing providers may benefit from emphasizing this testing’s comparative convenience and privacy compared with face-to-face testing in order to improve uptake, as well as alleviating concerns about the self-sampling process. There is a clear need for further research exploring in depth the perceptions and experiences of people who have accessed internet-based testing and for research on internet-based testing that explicitly gathers the views of populations that are at high risk of STIs. Trial Registration: PROSPERO CRD42019146938; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=146938 [J Med Internet Res 2020;22(8):e17667]