Mass spectrometry (MS) often requires vacuum conditions, which, while beneficial for analysis, can unpredictably alter sensitive samples. This study investigates the impact of prolonged vacuum exposure on the consistency and reliability of MS detection of thin films of acetaminophen using secondary ion mass spectrometry (SIMS). Under vacuum at room temperature, the mass spectrometry signal intensity decreased by approximately 81.5% over the duration of the experiment (42 h). Optical microscopy revealed that this decrease coincided with sublimation-induced sample loss of the acetaminophen. As a result, acetaminophen coverage across the substrate became heterogeneous, leading to increased relative standard deviation (RSD) in the SIMS signal over time. In contrast, under cryogenic conditions, neither signal degradation nor an increase in RSD was observed. Additionally, a comparison with atmospheric pressure mass spectrometry revealed that, in the absence of vacuum, signal intensity remained more stable over time. These findings highlight the potential drawbacks of vacuum exposure for volatile standards and emphasize the importance of testing vacuum effects prior to analysis. If vacuum is necessary, cryogenic conditions should be considered to mitigate sample degradation. While these effects were observed for a mass spectrometry technique, they are also applicable to any type of vacuum-based methodology where the samples might be prone to sublimation.