A highly sensitive and selective reversed-phase high performance liquid chromatography (RP-HPLC) with fluorescence detection for topotecan (SK&F 104864A) trace analysis is presented. Topotecan is the prototype of a novel class of antitumor agents, which exert their activities exclusively by inhibition of cellular topoisomerase I. The analytical method is based on an efficient HPLC separation using a Zorbax® StableBond® narrow bore (150 × 2.1 mm i.d.) C8column (particle size 5 mm). A secondary-retention phenomenon was noticed during the studies, however, and the problem was solved by adding 0.1% (v/v) triethylamine. Zorbax® StableBond® columns represent the new class of silane-modified silica, and have proved to be stable at least four months with continuously analyzed samples containing bleach and hydrogen peroxide. Column effluents were monitored via fluorescence with the excitation wavelength set at 225 nm and the emission wavelength at 520 nm. The limit of quantification (LOQ) for topotecan was 0.2 ng/mL. Linear response was observed for concentration of topotecan from 0.2 to 20 ng/mL. The method not only provided sensitive, precise and accurate support to photolysis, biodegradation and deactivation treatment system studies but also reduced the organic solvent waste by two-thirds as compared to conventional HPLC columns (4.6 mm i.d.).