Diazacon (20,25-diazacholesterol) is a cholesterol mimic that inhibits cholesterol production and blocks steroid hormone formation. It is also used as an additive to bird feed as contraceptive to control population of various birds. LC/MS compatible method was developed on Primesep B2 column. Method can be used for quantitation of diazacon in biofluids and bird feeds. It is analyzed in reversed-phase cation-exclusion mode on Primesep B2 mixed-mode HPLC column. Retention time is controlled by amount of acetonitrile and amount of buffer. Due to ion-exclusion mechanism buffer concentration has a reversed effect on retention – more buffer in the mobile phase provides longer retention. Reversed gradient of buffer is used to improve peak shape and facilitate faster elution. Method can be used for analysis of other hydrophobic basic compounds. Method is compatible with ELSD and LC/MS detection.