Neospora caninum is an obligate intracellular protozoan parasite of the phylum Alveolata (subphylum Apicomplexa) which has not been studied extensively in a biochemical context. N. caninum is a primary cause of reproductive disorders causing mummification and abortion not only in cattle but also in other small ruminant species resulting in a substantial economic impact on the livestock industry. In canids, which are the final hosts of N. caninum, clinical disease includes neuromuscular symptoms, ataxia, and ascending paralysis. Fatal outcomes of neosporosis have also been reported depending on the host species, age and immune status, however, its zoonotic potential is still uncertain. Therefore, N. caninum should be thoroughly investigated. Matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) and MS imaging (MSI) were used, combined with high‐performance liquid chromatography (HPLC) to investigate these intracellular parasites. The aim of this study was to identify molecular biomarkers for N. caninum tachyzoite‐infected host cells and to further clarify their functions. By atmospheric‐pressure scanning microprobe MALDI MS(I), sections of N. caninum‐infected and non‐infected host cell pellets were examined in order to determine potential markers. In vivo, N. caninum infects different types of nucleated cells, such as endothelial cells which represent a highly immunoreactive cell type. Therefore, primary bovine umbilical vein endothelial cells were here used as a suitable infection system. For comparison, the permanent MARC‐145 cell line was used as an additional, simplified in vitro cell culture model. HPLC‐tandem MS (HPLC‐MS/MS) experiments combined with database search were employed for structural verification of markers. The statistically relevant biomarkers found by MS and identified by HPLC‐MS/MS measurements were partly also found in infected monolayers. Marker signals were imaged in cell layers of N. caninum‐infected and non‐infected host cells at 5 µm lateral resolution.