The objective of this study was to develop a precise and accurate method to quantify cholesterol in turkey meat products using relative response factors, based on a modification of a previously published method for plant sterols determination. Validation was performed using neat solutions to determine linearity, precision, and accuracy. The method was linear in the concentration range considered (1–20 µg/mL, r2 ≥ 0.991). Precision and accuracy were within the acceptability guidelines of the U.S. Food and Drug Administration (FDA) for method validation (<20% relative standard deviation (RSD) at the lower limit of quantification (LLOQ) and <15% RSD for other standards). Turkey meat was spiked with cholesterol at two levels (low = 3 µg/mL and high = 18 µg/mL), either before or after saponification, to establish the recovery and matrix effects. Recovery ranged from 94% to 105%, with a mean value of 105% at the low spike level and 95% at the high spike level. No significant matrix effects were found (90% to 112% recovery). This method is reliable for the quantification of cholesterol in turkey meat products in the range 0.4–8 mg/g.