Single-molecule techniques are currently an essential tool to study conformational changes as well as the synthesis and folding of proteins. However, the preparation of suitable protein samples is often time-consuming and demanding. The rapid development of cell-free protein synthesis over the last few years opened new perspectives for fast and easy sample preparation, but this was not fully exploited until now. Here, we take a look at the advancements in sample preparation as well as in the development of technical approaches and analytical tools, which unavoidably lead to the combination of single-molecule techniques and cell-free protein synthesis. It is an ideal combination that can unlock the full potential of studying complex biological processes in the near future.