HIV pre-exposure prophylaxis (PrEP) is a preventative strategy that involves taking a daily, oral medication to reduce the risk of contracting HIV. Advancements in recent years may have changed the ways in which PrEP is discussed in public discourse, and the use of social media for openly discussing and disseminating PrEP-related promotion messages has been understudied. Thus, this study focuses on understanding the current discourse surrounding PrEP on Twitter. Through a quantitative content analysis, this study examined the messaging, sources, and sentiments and emotions expressed in PrEP-related tweets posted from April 2019 to April 2020, six months before and after the FDA approval of Descovy for PrEP. After the approval of Descovy, there was a significant decrease in tweets relating to barriers for using PrEP; further, informational tweets about PrEP significantly increased. However, a large proportion of tweets still discussed barriers (29.9%), suggesting that addressing barriers to PrEP remains a top priority for promoting PrEP uptake. A lack of tweets discussing individual attitudes, norms, and behavioral control about PrEP may suggest that the social media discourse is not covering the important aspects of behavioral determinants for PrEP adoption. Health communication researchers can focus on developing message strategies that target these theoretical constructs to promote the adoption of PrEP among key target populations.