A simple and novel method is proposed here for the first time to determine pKa values of chromogenic hydrophobic pH sensitive probes directly in nanospheres. pKa values can be obtained by measuring the pH response of the nanospheres (containing the probes and ion exchanger) followed by measuring the pH and Na+ responses of the nanospheres (containing solvatochromic dyes and ion exchanger). The pKa values of four chromoionophores were successfully determined. This method is in principle also applicable to characterize colorimetric probes in other water immiscible nanomaterials.