Introduction The aim of this retrospective study was to evaluate the preservation of biomarkers immunoreactivity on cytospins protected with polyethylene glycol (PEG). Methods In two independent cytopathology laboratories, immunocytochemical reactions were retrospectively evaluated on methanol‐fixed and PEG‐protected cytospins stored at room temperature (RT) for different time periods and compared with immunocytochemical reactions on corresponding baseline methanol‐fixed cytospins. Semi‐quantitatively assessed immunoreactivity, using scores from 0 to 3, was considered reduced if two sequential scores were lowered by at least one point. Results Immunocytochemical reactions for 40 biomarkers with membrane (10), cytoplasmic (22) and nuclear (8) localisation were performed on 921 slides prepared from 183 cytological samples. For the majority of biomarkers (29/37, 78%), immunoreactivity on PEG‐protected cytospins stored at RT remained unchanged in the first 12 months. Immunoreactivity for GFAP, p40 and hepatocyte antigen was monitored and remained unchanged for 1, 8 and 7 months, respectively. Partial or complete loss of immunoreactivity on PEG‐protected cytospins stored for less than 12 months was found on a single sample out of the total evaluated for CD3 (1/7), CD30 (1/4), CD45 (1/10), CK5/6 (1/7), MelanA (1/7) and vimentin (1/7), while more frequent changes of immunoreactivity were found for Ki67 (4/7) and p63 (2/7). Conclusion Immunoreactivity on cytospins protected with PEG and stored at RT is well‐preserved for at least 12 months for the majority of biomarkers. Cell morphology and immunoreactivity of biomarkers is well preserved for at least 12 months on methanol‐fixed cytospins protected with polyethylene glycol and stored at room temperature. Appropriate cytology control slides can be prepared for immunocytochemistry on cytology samples