This study focuses on the synthesis, characterization and application of p-tetranitrocalix[4]arene-appended silica-based material as a stationary phase in high-performance liquid chromatography for the simultaneous separation of some basic dyes such as methyl violet, methyl green and methylene blue. The effect of the pH of mobile phase on the retention, separation and selectivity of these compounds was investigated. The method was validated for system efficiency, linearity, accuracy, precision, limit of detection and quantification. The limits of detection were 0.1, 0.5 and 0.3 mg L⁻¹; whereas, the limits of quantification were observed to be 1, 1.2 and 0.9 mg L⁻¹ for methyl violet, methyl green and methylene blue, respectively. The separation efficiency of the p-tetranitrocalix[4]arene-appended silica-based column was also compared to that of the octadecylsilane column.