Determining the formation temperature of minerals using fluid inclusions is a crucial step in understanding rock-forming scenarios. Unfortunately, fluid inclusions in minerals formed at low temperature, such as gypsum, are commonly in a metastable monophase liquid state. To overcome this problem, ultra-short laser pulses can be used to induce vapor bubble nucleation, thus creating a stable two-phase fluid inclusion appropriate for subsequent measurements of the liquid-vapor homogenization temperature, Th. In this study we evaluate the applicability of Th data to accurately determine gypsum formation temperatures. We used fluid inclusions in synthetic gypsum crystals grown in the laboratory at different temperatures between 40 °C and 80 °C under atmospheric pressure conditions. We found an asymmetric distribution of the Th values, which are systematically lower than the actual crystal growth temperatures, Tg; this is due to (1) the effect of surface tension on liquid-vapor homogenization, and (2) plastic deformation of the inclusion walls due to internal tensile stress occurring in the metastable state of the inclusions. Based on this understanding, we have determined growth temperatures of natural giant gypsum crystals from Naica (Mexico), yielding 47 ± 1.5 °C for crystals grown in the Cave of Swords (120 m below surface) and 54.5 ± 2 °C for giant crystals grown in the Cave of Crystals (290 m below surface). These results support the earlier hypothesis that the population and the size of the Naica crystals were controlled by temperature. In addition, this experimental method opens a door to determining the growth temperature of minerals forming in low-temperature environments.