A few new psychoactive substances (NPS) that mimic the effects of controlled neuropsychiatric and illicit drugs have been forensically identified in the U.S. Wastewater-based epidemiology (WBE) can provide a comprehensive and more cost- and time-effective method of determining the prevalence of NPSs in communities. In this study, an analytical method capable of simultaneous determination of trace-level 40 NPS residues (synthetic opioids, synthetic cannabinoids, synthetic cathinones, piperazines, indole, and amphetamine) in wastewater was developed and validated. The developed analytical method was utilized to determine the occurrence of NPSs in four rural communities in southern Illinois. Nine NPSs (carfentanil, furanyl fentanyl, methoxyacetyl fentanyl, MAB-CHMINACA, methcathinone, 4-methyl pentedrone, 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MMMP), 1-(3-chlorophenyl) piperazine (mCPP), and 5-(2-Aminopropyl) Indole (5IT) were quantified. Methcathinone was the most frequently detected NPS (detection frequency, df = 100%) followed closely by the MMMP and mCPP (df = 91%). The mass loading of methcathinone, mCPP, and 5-IT using ammoniacal nitrogen-based population were up to 21.1 ± 1.3 mg/d/1000 people, 15.0 ± 0.5 mg/d/1000 people, and 9.75 ± 2.72 mg/d/1000 people, respectively. This is the first study to determine the occurrence of NPSs including synthetic opioids, synthetic cannabinoids, synthetic cathinones, and piperazines in the U.S. communities.