A reversed-phase ionpair chromatographic method with tetramethylene oxide as organic modifier has been developed for the simultaneous separation and detection of the sulfides NH2CSNH2, (NH4)2CS3, (NH4)2S, and NH4SCN. The optimized separation conditions were determined by means of a U7 (76) uniform design experiment, and tetramethylene oxide played an important role in adjusting the retention behavior of (NH4)2S and NH4SCN. The linearity of the calibration plots for the four components was investigated; correlation coefficients were from 0.9989–0.9999. The proposed method was successfully applied to the determination of NH2CSNH2, (NH4)2CS3, (NH4)2S, and NH4SCN in synthesis and isomerization samples.