In 2020 the world faced the pandemic of COVID‐19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS‐CoV‐2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID‐19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS‐CoV‐2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme‐linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients’ tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS‐ CoV‐2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS‐CoV‐ 2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS‐CoV‐2, even if the virus acquires novel mutations. © 2020, MDPI AG. All rights reserved.