Objective: To cross-culturally adapt and validate the Functional Assessment Scale for acute hamstring injury for professional Spanish-speaking soccer players. Design: Clinical measurement study. Cross-cultural adaptation was conducted following international recommendations. Indicators of validity, reliability and responsiveness are provided. Subjects: The Spanish version of the Functional Assessment Scale for acute hamstring injury scale was administered to 165 participants: 45 professional soccer players with acute hamstring muscle injury diagnosis, 40 healthy subjects, 40 individuals at-risk for a hamstring muscle injury and 40 patients with injuries of the lower limb other than hamstring muscle injury. Main measures: The Functional Assessment Scale for acute hamstring injury. Reference measures: Spanish version of the Quality of Life Short-Form 36 questionnaire (SF-36) and the Lower Limb Functional Index (LLFI). Results: Cronbach’s alpha (internal consistency) for the Spanish version of the Functional Assessment Scale for acute hamstring injury scale was >0.8. The intraclass correlation coefficient using the two-way random model (ICC_2,1) (test–retest) was 0.993 (95% confidence interval (CI): 0.991–0.995; P < 0.05). In the exploratory factor analysis, a one-factor solution explained 85% of the variance. Subjects with hamstring muscle injury scored significantly lower than the other groups in the Spanish version of the Functional Assessment Scale for acute hamstring injury scale ( P < 0.001). The Spanish version of the Functional Assessment Scale for acute hamstring injury scale score within the hamstring muscle injury group showed moderate and significant correlations with SF-36 physical components (Spearman’s r_s > 0.6; P < 0.001), and LLFI score at baseline ( r_s = 0.42; P < 0.01). The standard error of measurement (SEM) and minimum detectable change threshold (MDC_95%) were 2.6 and 7.2 points, respectively. The responsiveness indicators have an effect size of 3.62, and the standardized response mean is 3.24. Conclusion: The Spanish version of the Functional Assessment Scale for acute hamstring injury scale showed satisfactory psychometric properties. It can be considered a reliable and valid instrument to assess the functional impact of acute hamstring muscle injury in professional Spanish-speaking football players.