Abstract Introduction Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are common sexually transmitted infections (STIs) that are often asymptomatic, delaying diagnosis and treatment which can cause health consequences and increased transmission. The CDC suggests that self-collected (SC) samples (urine; vaginal, rectal, oropharyngeal swabs) are reasonable alternatives to provider-collected (PC) samples. However, only urine and vaginal swabs are commercially offered for within-clinic self-collection leaving a gap for (1)detecting extra-genital infections (common among LGBTQ+ community) and (2)improving access by allowing for at-home self-collection. The objective of this study was to elucidate analytical and pre-analytical variables associated with at-home SC STI testing with the intention of improving inclusion and access to sexual healthcare. Methods Paired PC/SC rectal (n = 164) and throat (n = 159) swabs were compared for CT/NG detection; reproducibly equivocal results (n = 3) were considered positive. Limit of detection (LoD) for CT/NG was performed by serial dilution to determine a concentration at the point of failure below which detection becomes unreliable (n = 20). Concentrations of 2–10× the LoD were used in subsequent experiments. Interference testing was performed to determine if hand contaminants (1% v/v) would impact assay performance (n = 5/contaminant). To evaluate shipping stability, samples were challenged using winter or summer simulation with temperature cycling designed to mimic extreme seasonal fluctuations. Additional experiments were performed on urine samples to evaluate the impact of underfilling/overfilling or removing the transport medium from the sample tube. CT/NG detection was performed using the Hologic Aptima Combo 2 Nucleic Acid Amplification Assay. Results Relative to provider collected samples, rectal swabs demonstrated a positive agreement of 95.5% for CT (n = 22 (13.4%) positive PC) and 100% for NG (n = 9 (5.5%) positive PC) with an overall agreement of 97.5% for CT and 98.1% for NG. Throat samples demonstrated a positive agreement of 100% for both CT and NG (CT: n = 2 (1.3%) positive PC; NG: n = 11 (6.9%) positive PC) with an overall agreement of 100% for CT and 96.8% for NG. Although there was one false negative rectal SC relative to PC, almost all swab/organism combinations had positive results by SC that were negative by PC: 3+ SC for rectal CT; 3+ SC for rectal NG; 5+ SC for throat NG. LoDs were 0.03–0.06 IFU/mL for CT and 0.06–0.5 CFU/mL for GC using urine or vaginal swabs. Hand contaminants did not interfere with CT/NG detection, and samples demonstrated ≥ 95% agreement after winter and summer shipping stability challenges. Urine results were unaffected by underfilling (≥0.5 mL urine), overfilling (≤4.5 mL urine), or removal of transport medium (≥0.5 mL buffer volume). Conclusions Self-collection of extra-genital swabs for CT/NG detection may offer better sensitivity than physician collected samples without compromising the analytical prowess of the assay. Home collected STI specimens are a viable option for improving access to STI screening and offer a non-stigmatizing approach to sexual health.