BACKGROUND: Little empirical evidence exists on the effectiveness of using Twitter as a two-way communication tool for public health practice, such as Twitter chats. OBJECTIVE: We analyzed whether Twitter chats facilitate engagement in two-way communications between public health entities and their audience. We also describe how to measure two-way communications, incoming and outgoing mentions, between users in a protocol using free and publicly available tools (Symplur, OpenRefine, and Gephi). METHODS: We used a mixed-methods approach, social network analysis, and content analysis. The study population comprised individuals and organizations participating or who were mentioned in the first #LiveFitNOLA chat, during a 75-min period on March 5, 2015, from 12:00 PM to 1:15 PM Central Time. We assessed audience engagement in two-way communications with two metrics: engagement ratio and return on engagement (ROE). RESULTS: The #LiveFitNOLA chat had 744 tweets and 66 participants with an average of 11 tweets per participant. The resulting network had 134 network members and 474 engagements. The engagement ratios and ROEs for the #LiveFitNOLA organizers were 1:1, 40% (13/32) (@TulanePRC) and 2:1, -40% (-25/63) (@FitNOLA). Content analysis showed information sharing (63.9%, 314/491) and health information (27.9%, 137/491) as the most salient theme and sub-theme, respectively. CONCLUSIONS: Our findings suggest Twitter chats facilitate audience engagement in two-way communications between public health entities and their audience. The #LiveFitNOLA organizers' engagement ratios and ROEs indicated a moderate level of engagement with their audience. The practical significance of the engagement ratio and ROE depends on the audience, context, scope, scale, and goal of a Twitter chat or other organized hashtag-based communications on Twitter.