Introduction: in Nigeria, it was estimated that 1.9 million people were living with HIV of which 130,000 people were newly infected with HIV. HIV self-testing would potentially increase access to HIV testing for people to know their status, get diagnosed, and initiate treatment as soon as possible. Our study aims to assess the knowledge of HIV Self-Testing (HIVST) and the acceptability of this youth-friendly approach among students in southwest Nigeria online. Methods: a cross-sectional study was conducted among bona fide undergraduate students (2019/2020 session) of two popular tertiary institutions in southwest Nigeria. An online standardized self-administered questionnaire was administered using Google Forms. Microsoft Excel and IBM SPSS statistics were used for tabulation and statistical data analysis. The Chi-Square test was conducted using a P value of 0.05 to determine the level of significance. Results: of the 155 students that participated in the study, 82 (52.9%) were male. Most of the respondents (65.2%) were studying medicine and other health-related courses. The mean knowledge of HIVST among respondents was slightly above average. Respondents studying medical and other health-related courses showed a slightly better level of knowledge than others although not statistically significant (P = 0.222). 76.1% of respondents had never used the HIVST option before and 62.6% are willing to use it sometimes in the future. Conclusion: to achieve the UNAIDS 95-95-95 fast-track targets in Nigeria by 2030, there is a need to promote sexual and reproductive health education and increase awareness and accessibility of HIVST to youths.