Context: Limited research reveals that the use of different soft tissue mobilization techniques increases tissue mobility in different regions of the body. Objective: The purpose of this study was to determine whether there is a difference between administering instrument-assisted soft tissue mobilization (IASTM) and therapeutic cupping (TC) on hamstring tightness. Design: Subjects attended one session wherein treatment and leg order were randomized before attending the session. A statistical analysis was completed using a 2 (intervention) × 2 (time) repeated-measures analysis of variance at α level ≤ .05. Participants: Thirty-three subjects between the age of 18–35 years old with bilateral hamstring tightness participated in this study. Interventions: The IASTM and TC were administered on different legs for 5 minutes and over the entire area of the hamstring muscles. One TC was moved over the entire treatment area in a similar fashion as the IASTM. Main Outcome Measures: The intervention measurements included soreness numeric rating scale, Sit-n-Reach (single leg for side being tested), goniometric measurement for straight-leg hip-flexion motion, and superficial skin temperature. The timeline for data collection included: (1) intervention measurements for the first randomized leg, (2) 5-minute treatment with the first intervention treatment, (3) intervention measurements repeated for postintervention outcomes, and (4) repeat the same steps for 1 to 3 with the contralateral leg and the other intervention. Results: There was a main effect over time for Sit-n-Reach, measurement (pre-IASTM—29.50 [8.54], post-IASTM—32.11 [8.31] and pre-TC—29.67 [8.21], post-TC—32.05 [8.25]) and goniometric measurement (pre-IASTM—83.45 [13.86], post-IASTM—92.73 [13.20] and pre-TC—83.76 [11.97], post-TC—93.67 [12.15]; P < .05). Conclusion: Both IASTM and TC impacted hamstring mobility during a single treatment using only an instrument-assisted soft tissue mobilization technique without any additional therapeutic intervention.