An HPLC method for determination of related substances in temozolomide drug substance was developed. Particular attention was paid to the stability studies due to the fact that temozolomide is unstable in a solution and quickly decomposes to its main degradation product 5-amino-4-imidazolecarboxamide (AIC). A mixture of diluted acetic acid and acetonitrile (4:1, v/v) as a diluent guaranteed lowering the decomposition of temozolomide in the solution. As it is not practically possible to fully eliminate the decomposition of temozolomide during an analysis, the mathematical correction of the results was proposed which allows to analyse almost five times more samples per week, comparing to the procedure without the application of the correction. The accuracy of the correction procedure was proved by investigating the recovery of AIC spiked to temozolomide solutions at different levels. Recoveries equalled 90–108% for AIC concentrations contained in the range of 0.30–1.80 μg ml−1. The developed method was validated according to the current guidelines, proving the suitability of the method for its intended purpose.