Electroporation is a method used for local cancer therapy in a form of electrochemotherapy or electroablation [1]. The mechanism of electrochemotherapy is triggered by the process of reversible electroporation; when high intensity electric field increase transmembrane potential of affected cells to a critical value. This triggers the formation of hydrophilic pores in the cellular membrane and act as a bridge between cytosol and extracellular matrix for various hydrophilic substances such as calcium ions or anticancer drug bleomycin [2]. If the transmembrane potential is much higher than the electroporation threshold, then the process of irreversible electroporation occurs, hence triggering anticancer treatment of electroablation. The use of such treatment initiate affected cell death as a result of triggered irreversible changes in cell plasma membrane [3]. Both therapies share similarities with another method that has been applied in cancer therapy – radiotherapy. The main mechanism of those treatments is based on the generation of reactive oxygen species (ROS) in the targeted tissue, resulting in multiple genomic DNA breaks that may lead to cell death. It is known that irradiated cells at lethal doses does secrete signaling molecules that affect adjacent unirradiated cells. This phenomenon is termed as the "Bystander effect" [4]. Although, the "Bystander effect" is well-known in radiotherapy, it has not been researched in the process of electrochemotherapy. According to our resent publication, intracellular electrotransfer of bleomycin into the targeted cells result in decrease of viability on the directly unaffected adjacent cells. Controversially, irreversible electroporation on the targeted cells has a positive impact to the viability of directly unaffected adjacent cells [5]. Further investigation of these inverse phenomena of Bystander effect after bleomycin electrotransfer and irreversible electroporation led to the combination of these processes. Therefore, the aim of this study is to evaluate the "Bystander effect" when combining CaCl2 electrotransfer, irreversible electroporation and electrotransfer of anticancer drug bleomycin. Mouse breast cancer cell line (4T1) was used for the presented experiments. Electric fields were generated with BTX T820 system. Stainless steel plate electrodes with 2 mm gap were used. Laboratory-made electroporation buffer (Na2HPO4 5.59 mM, NaH2PO4 3.00 mM, MgCl2 1.73 mM, sucrose 242.2 mM) at conductivity of 0.1 S/m was used. To obtain CaCl2 and bleomycin electrotransfer one 100 s duration and 1400 V/cm amplitude pulse was applied. In the case of irreversible electroporation the amplitude was raised 2 times to 2800 V/cm. The cells after electroporation were incubated in a 24-well plate for 10 minutes, then a 0.2 ml RPMI growth medium was added and incubated for 48 hours. Afterwards the media was removed and put on untreated cells to measure the formation of colonies. The results showed that bystander effect is present when media from effected cells is transferred on the unaffected cells. Bleomycin electrotransfer triggered Bystander effect diminished the growth of directly unaffected cells. Similar results were obtained after calcium electrotransfer. An inverse Bystander effect was obtained after application of irreversible electroporation. Nevertheless, the effect is medium dependent. When the media was diluted twice all of the Bystander effects were not present. However, the combination of two bystander effects by adding 50 % of media from cells after bleomycin electrotransfer and 50 % of media from cells after irreversible electroporation had a highly negative impact on cell viability. Similar trends are observed when combining bystander effect after bleomycin electroporation and calcium electroporation