Abstract The separation of the two regioisomeric derivatives of 1 H ‐1,5‐benzodiazepine yielded from the reaction of 1,2‐diamino‐4‐methylbenzene with 4‐substituted acetophenones was performed by reversed phase high performance liquid chromatography, and the absorption spectra of the separated isomers have been determined for three isomer pairs which have been obtained starting from acetophenones with substituents of different electronegativity. The isomer ratio then could be estimated as well from the ratio of the peak areas as from the absorption spectra. They agree well with the known ratio determined from nmr intensities.