Sulfonamides (SAs) are one of the most frequently used antibiotics. SAs have been found in various environmental compartments. If SAs are not degraded in the environment, they can affect bacteria by their antibiotic properties and contribute to bacterial antibiotic resistance. Therefore, the biodegradability of 11 SAs (sulfanilamide, sulfaguanidine monohydrate, sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethazine, sulfamethoxazole, and sulfadimethoxine) was studied. For this purpose, the Closed Bottle Test (CBT, OECD 301D) was performed, which includes a toxicity control. In order to monitor the environmental fate of the parent compound and to check for transformation products, a simple, efficient, and reliable HPLC-UV method for the simultaneous determination of these SAs has been developed. Acetonitrile and water (with 0.1% formic acid) were used as mobile phase solvents for gradient elution. The method was validated in terms of precision, detection and quantitation limits, selectivity, and analytical solution stability. In the CBT, none of these SAs was readily biodegradable. The HPLC-UV analysis confirmed that no degradation of any SA took place. In the toxicity control, these SAs showed no toxic effect in the used concentration of environmental bacteria applied in the test.