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In this paper, we proposed an oocyte orientation selection method based on the minimum strain position in the penetration process in order to maximize the developmental potential of the operated oocytes. Considering the symmetry of an oocyte during the penetration process, we analyzed only the intracellular strain of the lower half of the oocyte (from 3 o’clock to 9 o’clock). Firstly, the strain distribution in the penetration process was calculated using the finite element analysis method. The influences of penetration force, zona pellucida thickness, oocyte radius, Young’s modulus of the cytoplasm, and zona pellucida on the strain distribution were studied. The simulation results showed that the strain values at the 4 o’clock and 9 o’clock areas were significantly smaller than those at the 5 o’clock, 6 o’clock, 7 o’clock, and 8 o’clock areas. Secondly, the experimental strain distribution during penetration was evaluated for the first time using an optical flow method. The experimental strain distribution was strongly positively correlated to the aforementioned simulated results. Finally, the developmental potential of the penetrated porcine oocyte with different orientations was evaluated using the cleavage rate (48 h after penetration and parthenogenetic activation). The cleavage rate was strongly negatively correlated to the intracellular strain. We inferred that a smaller strain on the polar body areas in the penetration process caused less potential damage, which leads to a higher developmental potential of the penetrated oocyte. The optimized oocyte orientations, with the polar body at the 4 o’clock and 9 o’clock areas, are determined based on the minimum strain position in the penetration process.