Sexually transmitted infections (STIs) are a major global cause of acute illness, leading to serious complications (e.g. infertility and long term disability). Over 30 different bacteria, viruses and parasites are sexually transmitted, with Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and human papillomavirus being the most prevalent curable causative agents. Although Mycoplasma genitalium is frequently (co-)detected as well, the clinical importance is less clear. STI prevention and treatment focus on reducing the prevalence by interrupting transmission, reducing the duration of infection and preventing complications. Early detection of (a) symptomatic infections have a crucial role to play in reaching these objectives. Multiple molecular diagnostic tools have been developed to detect a variety of causative agents, most of them detecting only one pathogen at a time and in different sample types. A first goal of this PhD is to improve the accessibility of STI testing, in particular for hard-to-reach populations, by optimizing the sample collection. This will result in diagnosis and treatment to be available more quickly, thereby curbing the clinical impact. A multiplex diagnostic approach may be appropriate, as STIs are frequently multi-pathogenic and in part asymptomatic. However, a “pan-approach” without sufficient knowledge of the clinical value of each target is to be avoided, as it can lead to overtreatment and unnecessary patient anxiety.