Aims of the study were (1) to establish a method for quantification of chlorhexidine (CHX) in small volumes and (2) to determine CHX release from differently concentrated CHX-containing preparations, varnishes, and a CHX gel applied on artificial fissures. CHX determination was conducted in a microplate reader using polystyrene wells. The reduced intensity of fluorescence of the microplates was used for CHX quantification. For verification of the technique, intra- and inter-assay coefficients of variation were calculated for graded series of CHX concentrations, and the lower limit of quantification (LLOQ) was determined. Additionally, artificial fissures were prepared in 50 bovine enamel samples, divided into five groups (A-E, n=10) and stored in distilled water (7 days); A: CHX-varnish EC40; B: CHX-varnish Cervitec; C: CHX-gel Chlorhexamed; D: negative control, no CHX application; and E: CXH-diacetate standard (E1, n=5) or CHX-digluconate (E2, n=5) in the solution. The specimens were brushed daily, and CHX in the solution was measured. The method showed intra- and inter-assay coefficients of variation of <10 and <20%, respectively; LLOQ was 0.91-1.22 nmol/well. The cumulative CHX release (mean+/-SD) during the 7 days was: EC40 (217.2+/-41.8 nmol), CHX-gel (31.3+/-8.5 nmol), Cervitec (18.6+/-1.7 nmol). Groups A-C revealed a significantly higher CHX release than group D and a continuous CHX-release with the highest increase from day 0 to 7 for EC40 and the lowest for Chlorhexamed. The new method is a reliable tool to quantify CHX in small volumes. Both tested varnishes demonstrate prolonged and higher CHX release from artificial fissures than the CHX-gel tested.