Miniaturization of cell-based assays enables the analysis of secreted compounds from low cell numbers down to a single cell. Droplet microfluidics is a well-established tool for high-throughput single-cell analysis. Nevertheless, the integration of label-free bioanalytical techniques like mass spectrometry is still ongoing. For example, without additional separation steps, droplet-enclosed cells do not survive the analysis. Cell separation techniques for droplets have been reported, but could not yet be coupled to mass spectrometric analysis. Here, we present a simple approach for high-throughput cell separation in parallel in nanoliter droplets and demonstrate that it can be used for qualitative analysis of protein secretion by the yeast Komagataella phaffii. Using a custom-made droplet spotter, we generated an array of 200 droplets of nanoliter volumes on a glass plate, each containing approximately 500 cells. After cultivation for 24 h, a second plate was placed above the droplet array and brought in contact with the droplets. All droplets were sampled in parallel by plate-based droplet splitting. The nanoliter samples of the supernatant could be interfaced with mass spectrometry and we were able to detect the protein brazzein (his-tagged, 7445 Da) in all but two droplets. Additionally, we show that the cells were viable after the cell separation and a sample from one spot could be transferred to a cultivation tube. An advantage of our protocol is that each cell suspension is directly linked to the analysis result by its position. Furthermore, we demonstrate that our method is capable of splitting around 6000 droplets in a few seconds. In the future, additional processing steps on a small scale, such as desalting and protein digestion, could be developed and will enable structural proteomics in nanoliter volumes.