Intraoperative determination of tumor boundaries reduces the volume of resected tissue while ensuring complete removal of malignant tissue without increasing the risk of recurrence. The existing approaches, including intraoperative histology or tomography, are highly effective, but they extend the time of the surgery. Therefore, real-time analysis methods are a particularly interesting avenue. One such solution involves building a profile using ambient ionization mass spectrometry. The molecular profile of the examined tissue is compared with a reference database of healthy and pathologically altered tissues. For many human organs, samples for this database can only be obtained from autopsy material. However, due to the unavoidable gap between the moment of death and sample collection, tissue degradation can change the picture of pathological process. This experimental quantitative study investigated the stability of polar lipid profiles in the liver and brain tissues of healthy BALB/c mice (n = 23) during the early postmortem period (0–72 hours). It has been shown that lysolipids and free fatty acids correlate with the postmortem interval (r > 0.6 overall; r = 0.75 for FA 20:4 and FA 22:6); therefore, they cannot be used as molecular markers in diagnostic models based on autopsy samples. At the same time, the profile of phospholipids in tissue cell membranes remains largely unchanged in the early postmortem period, which preserves their value as biomarkers detectable in both biopsy and autopsy specimens.