Inclusions in Ti investment castings are generally known to have detrimental effects on the performance of the castings. However, actual inclusions are infrequent and hard to be located. As a result, it is extremely difficult to obtain sufficient amount of fatigue test specimens of titanium investment castings having inclusions in the gage section. Thus, in-depth research of the adverse influence of inclusions is also hindered. To address this problem, a new casting methodology of specimens containing hard alpha inclusions was developed in this study. To guarantee successful introduction of an inclusion and casting, a carefully designed mold with 8 legs and a special tool were employed. After solidification, castings were cut, and X-ray radiography determined that the inclusions were successfully incorporated into the castings. The castings were further prepared to obtain multiple test specimens and they were fatigue-tested consecutively. Fractography analysis confirmed that fatigue cracks initiated at the hard alpha inclusion. In a nonlinear regression model, the fatigue life can be modeled as an exponential function with a negative exponent of the cross-sectional area of an inclusion. The fatigue life of Ti specimens containing inclusions is inversely proportional to the cross-sectional area of an inclusion.