We prepared a series of low-molecular-mass fluorescent ampholytes with narrow p/ range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent p/ markers, were examined. The p/ values of new fluorescein-based p/ markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass p/ markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.