The necessity of nicotine analysis in blood plasma is increasing along with the increased number of smokers and nicotine poisoning cases. One of the analytical methods for nicotine is using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detector because it has been commonly owned by instance in Indonesia. To guarantee accuracy, an analytical method can be used, and it must be validated. This research was the purpose of finding out the validity of the nicotine analysis method in human blood plasma (in vitro) using HPLC with UV detection. Blood plasma samples remained treated with centrifugation procedure by protein denaturation method using acetonitrile. The compounds were analyzed using methanol and buffer acetate 0.01 M (pH 5) 85:15 v/v as a mobile phase on an octadecylsilane column 250 mm, with UV detection at 254 and 260 nm, and flow rate 0.6 mL/minute. Parameter of analytical methods that were validated includes selectivity, accuracy, precision, repeatability, linearity, limit of detection (LoD), limit of quantification (LoQ), and system suitability. According to the result, the selectivity was 2.479, repeatability expressed by its variation coefficient = 0.701%, linearity at range 5–22 μg/mL expressed by coefficient correlation (r) = 0.996. Based on the chromatogram’s area under a curve, the LoD value was found 2.021 μg/mL, LoQ value was 6.737 μg/mL, the accurate percentage was 112.49 to 114.12%, and precision (% CV) was 2.15 to 3.95%. The system suitability from retention time and chromatogram’s area under curve showed % CV 0.70 and 1.64%. According to the experiment result, all parameters meet the requirements of validation criteria.
