Site specific modification of polypeptides with unnatural aminoacids (UAAs) is invaluable for exploring the details of their functioning and for endowing these molecules with novel functionalities. In particular, fluorescent spectroscopy that can provide information about protein structural dynamics relies on site-selective incorporation of at least two probes into a polypeptide chain. Presence of multiple reactive side chains in the polypeptides precludes their site-selective labelling. Co-translational incorporation of UAAs is complicated by the fact that dyes larger than Bodipy FL are incorporated very inefficiently. This can be overcome by incorporating of UAAs containing bioorthogonal functionalities with their subsequent derivatization. The most common approach for the incorporation of UAAs into polypeptide sequence is via codons orthogonal to translation machinery. These are either naturally occurring stop codons or artificially designed four-base codons. Although use of these methods both separately and in combination has been demonstrated, they severely compromise protein yields.We developed approach for effective protein modification in the cell-free protein synthesis system based on eukaryotic L.tarentolae organism. Here N-terminus of a protein is effectively labelled with a dye as a result of Ssp DnaB-mediated protein trans-splicing activity. Incorporation of UAA containing highly reactive bioorthogonal group (bicycle[6.1.0]nonyne) is achieved in response to the amber stop codon via use of M.mazei aminoacyl-tRNA transferase/tRNA pair. Although being semi flexible due to the constraints where one of the dies can be placed, this approach is advantageous over the use of two or more orthogonal codons since it does not significantly affect protein yield. Also because of this a present combined approach can be used in practically in any cell-free system or even in vivo.