Background: HIV-ASSIST is a free, continuously updated, clinically validated online algorithm tool that synthesizes participant- and virus-specific characteristics and provides ART decision support based on the goals of maximizing viral suppression and tolerability. The aim of this study was to analyze the concordance of clinicians’ ART preferences with HIV-ASSIST recommendations and the influencing factors. Methods: We conducted a cross-sectional retrospective cohort study using electronic medical records of people with HIV (PWH) followed in the Infectious Diseases and Clinical Microbiology Department of Health Sciences University Izmir Tepecik Training and Research Hospital. The concordance between prescribed ART and HIV-ASSIST recommendations was evaluated. Results: The study included 101 participants (92 male, 91.1%), median age was 35 (20–67), and 24.8% of participants were treatment-experienced. The concordance between prescribed ART and HIV-ASSIST recommendations was 90.1% (absolute concordance 60.4%). The concordance rate was 89.5% (absolute concordance rate was 64.5%) in treatment-naive participants; 92% (absolute concordance rate was 48%) in treatment-experienced participants. Factors that were associated with discordance in the multivariate analysis were co-trimoxazole prophylaxis and hyperlipidemia. Conclusion: The recommendations of the HIV-ASSIST algorithm were found to be highly concordant with the ART prescriptions of our clinicians.