The paper discusses some of the problems associated with the preparation and reversed-phase HPLC analysis of sirolimus samples in mixtures of water with methanol or acetonitrile with different content of the latter. It was shown that when the sirolimus samples were evaporated to dryness in polypropylene tubes, almost quantitative irreversible adsorption of the substance occurred. The loss of sirolimus can be avoided by using glass tubes for sample preparation when a complete removal of a solvent is necessary. Preparation of sirolimus samples in water–methanol or water-acetonitrile mixtures can lead to a gradual adsorption of the substance on the walls of a glass vial in case of a low organic solvent content. It was shown that a reconstitution solution containing about 50% of an organic solvent should be used to analyze sirolimus by the RP-HPLC method, since the analysis of samples dissolved in pure acetonitrile or methanol is limited by the amount of sample injected due to broadening or distortion of the chromatographic peak.