Abstract: AimTo establish a new and simple flow injection method for the rapid determination of acetylspiramycin (ASPM). MethodsASPM was determined by chemiluminescence (CL) method combined with flow injection (FI) technology, which was based on the inhibitive effect of ASPM on the chemiluminescence reaction of the luminol-K₃Fe(CN)6 system. ResultsThe decrease of chemiluminescence intensity was proportional to the logarithm of ASPM concentration (0.1-100) μg·L^-1, the detection limit was 40 ng·L^-1 (3σ). The whole process, including sampling and washing, could be completed in 0.5 min with a RSD less than 3.0% (n=5). ConclusionThe FI-CL method is of both high sensitivity and good selectivity giving a throughput of 120 h^-1. The proposed method was applied successfully to the determination of ASPM in pharmaceutical preparations and human urine without any pre-treatment. It was found that the ASPM concentration reached its maximum after being orally administrated for two hours.