A highly sensitive and specific fluorimetric method was developed for the determination of nalidixic acid. The proposed method involves the formation of a ternary complex with Tb3+in the presence of hexamine. The different experimental parameters were carefully studied and incorporated into the procedure. Under the described conditions, the proposed method is applicable over the concentration range of 0.1 – 2.4 μg ml−1with a detection limit (S/N = 2) of 2 ng ml−1. The recoveries from spiked urine and plasma were 101.40 and 96.98 percent, respectively.