This paper aims at the characterization of acid–base properties of nucleoside analogs using a pH-gradient flow injection -diode array detector (FI-DAD) system and further data analysis with multivariate curve resolution. Drugs selected comprise nucleoside reverse transcriptase inhibitors (NRTIs) extensively used in AIDS treatment, including zidovudine (AZT), didanosine (ddl), stavudine (d4T) and zalcitabine (ddC). The FI system consists of a two-channel manifold in which the sample injection into an acid carrier is responsible of the generation of a pH gradient. A first step is focused on an accurate calibration of the pH-gradient shape inside the FI peak using nucleoside analogs of known pK a values as standard compounds. The pH gradient in the FI peak is estimated from the concentration profiles of standard compounds. In a second step, the pH-gradient profile is exploited for a rapid estimation of unknown pK a values of test drugs. Various approaches for calculating pK a values are evaluated using the peak front or peak tail gradient ranges. As a result, overall errors in pK a estimations are lower than 2.5%.