Abstract Background: Trastuzumab is the treatment for HER2-positive breast cancer in the metastatic, adjuvant, and neoadjuvant settings. Trastuzumab is available in intravenous (i.v.) and subcutaneous (s.c.) application form. PrefHer study published in 2013 described patients' preference for use of subcutaneous trastuzumab. Our aim was to find out and describe if any preference exists in real clinical practice in the Czech Republic. Objectives: To analyse reasons for preference of s.c. or i.v. application of trastuzumab in patients with HER2-positive breast cancer in real clinical setting in the Czech Republic. Methods: A questionnaire based data collection from patients treated in Comprehensive Cancer Centres between January 2015 and July 2015. Results: We received 429 questionnaires from patients. Data analysis has been conducted in 301 (70.2%) patients' questionnaires after quality control for questionnaires completeness. Out of the 301 patients there were 151 who had ≥ 11 i.v. applications, 137 patients with 3-10 i.v. applications and 13 patients with only 2 i.v. applications. Majority of patients were treated in adjuvant setting (62.8%, n=189), 21.9% (n=66) received neoadjuvant treatment and 15.3% (n=46) patients were treated for advanced disease. Only 33 patients had experience with ≥ 11 s.c. applications, 222 patients with 3-10 s.c. applications and 46 patients with 2 s.c. applications. Subcutaneous form was given in 74.8% (n=225) in adjuvant setting, in 20.3% (n=61) in advanced setting and in 5.0% (n=15) in neoadjuvant setting. Pain related to trastuzumab application was assessed – 52.2% (n=157) patients stated that s.c. application is less painful, 34.2% (n=103) did not see difference and 13.6% (n=41) patients felt that i.v. form is less painful. When assessing site reactions majority (62.4%, n=184) of patients did not see difference between application forms, 20.0% (n=59) patients preferred s.c. form and 17.6% (n=52) preferred i.v. form. Lower anxiety related to trastuzumab application was reported with s.c. form (46.0%, n=137). Only 6.0% (n=18) patients reported lower anxiety with i.v. application and 48.0% (n=143) did not see any difference. Vast majority (92.7%, n=279) patients described s.c. form as more comfortable, 4.7% (n=14) patients did not see difference and only 2.7% (n=8) described i.v. form as more comfortable. Overall 95.0% (n=286) patients preferred s.c. application form of trastuzumab. The main reasons are time savings (86.7%, n=261) and better comfort (71.8%, n=216). Only 10 patients (3.3%) favoured i.v. application form. The main reason for i.v. preference is lower pain and lower incidence of complications (2.0%, n=6). The remaining 5 patients (1.7%) had no preference of any application form. Conclusion: Patients treated in Comprehensive Cancer Centres in the Czech Republic prefer subcutaneous application form of trastuzumab. The main reasons for their preference are time savings and better application comfort. Citation Format: Petrakova K, Melichar B, Bortlicek Z, Hejduk K. Preference of trastuzumab administration route (intravenous or subcutaneous) in patients in the Czech Republic. Cross-sectional study on 429 patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-12.