<b><i>Introduction:</i></b> The objective of this study was to construct gestational age (GA) based reference values for left ventricle (LV) longitudinal strain in normal fetuses, between 24 and 37 weeks’ gestation, assessing its feasibility and reproducibility with automated cardiac motion quantification software (aCMQ-QLab), which is widely used in postnatal echocardiography. <b><i>Methods:</i></b> This prospective study included healthy gravid women with singleton pregnancies and no evidence of fetal structural cardiovascular disease. Fetal echocardiographies were performed between 24 and 37 GA. 2D four-chamber view clips were recorded and LV longitudinal strain was analyzed offline. Intra- and interobserver reproducibility between 2 independent observers was evaluated by intraclass correlation coefficients (ICC) and Bland-Altman scatterplots. Regression analysis was used to determine GA adjusted reference ranges and construct nomograms. <b><i>Results:</i></b> LV longitudinal strain measurements were feasible in 95.4% of acquisitions. 435 clips were obtained. Intra- and interobserver ICC were 0.998 (95% CI 0.997–0.999) and 0.991 (95% CI 0.984–0.995), respectively. The global longitudinal strain and the middle and apical LV segments showed progressive decline as GA advanced, whereas the basal segments remained stable. <b><i>Conclusions:</i></b> Assessment of LV longitudinal strain by aCMQ-QLab is feasible, reproducible, and within normal ranges. Our results offer more information regarding fetal cardiac function assessment with 2D speckle tracking techniques, aiding in the introduction of this software into research practice, encouraging the realization of more studies, and probably helping in its future use in clinical practice, allowing longitudinal surveillance of strain without intervendor variability and aiding in follow-up of fetal cardiac conditions before and after birth, as it is the most commonly used software postnatally.