In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required.