The determination of ethyl [4-oxo-8-(3-chlorophenyl)-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl]acetate (ETTA), a new anticancer prodrug, using adsorptive stripping voltammetry (AdSV) was described for the first time. This method is based on adsorptive/reductive behaviour of ETTA at an in situ plated bismuth film electrode (BiFE) as a sensor. A number of experimental variables (e.g., a composition and pH of the supporting electrolyte, the conditions of bismuth film deposition, an accumulation potential and time, the scan rate, etc.) were thoroughly studied in order to achieve a high sensitivity. Experimental results under optimized conditions revealed an excellent linear correlation between the monitored voltammetric peak current and the ETTA concentration in the range of 2–50 μg L−1 following an accumulation time of 300 s. The limit of detection (LOD) for ETTA following 300 s of an accumulation time was 0.4 μg L−1. The proposed facile, sensitive and inexpensive method was successfully applied to the determination of ETTA in serum. The investigated prodrug was extracted from serum using SPE method.