Irinotecan is an anticancer drug for which significant benefits from personalised dosing are expected. Quick procedures are therefore essential for monitoring irinotecan in treated patients. The objective of this work was to develop and validate a rapid and simple visible spectrophotometric method for quantitative determination of irinotecan in pharmaceutical dosage forms and to further investigate its usefulness for irinotecan analysis in plasma. Based on the shift of the irinotecan 355/368 nm-peak at very low pH (0.2) to 400 nm, we established a linear relationship between absorbance at 400 nm and irinotecan concentration in dilutions of an irinotecan solution for injection (R² ≥ 0.999) and in plasma containing irinotecan (R² ≥ 0.995). Background absorbance correction at 455 nm was essential to minimise background interference, solely in plasma samples. We fully validated the assay for quality control of the irinotecan solution in the injection dosage form: the standard curve was linear over the concentration range of 0.90 to at least 37.00 μg ml⁻¹. The CV% on all quality control levels was determined to be ≤5.81% for repeatability and ≤6.62% for intermediate precision. Recovery was between 96.5% and 101.9%. Upon comparison with the LC/UV method, we demonstrated very good agreement and acceptable bias between the two methods (slope 0.973, y-intercept 0.0064). Similarly, the technical parameters of the assay in plasma satisfied international guidelines for method validation: the useful analytical range was determined to be between 0.93 and at least 10.00 μg ml⁻¹. This is suitable for quantifying irinotecan in the plasma of treated patients, in the upper region of its therapeutic window, to decide whether dose adjustment is required. Repeatability and intermediate precision (CV%) were within 4.49% and 9.91%, respectively. Recovery was between 96.3% and 103.8%. There was a lack of significant interference by mild hemolysis or by icterus. Irinotecan extraction efficiency from plasma was within 77.9–68.5%. Our results indicated that the proposed method allows quantitative determination of irinotecan plasma levels with acceptable analytical characteristics. The advantages of the proposed method in both matrices, in terms of specificity, rapidity, simplicity, environmental impact and cost effectiveness, are discussed.