Cardiorespiratory fitness is a fundamental determinant of both health outcomes and athletic performance, and is most objectively quantified through maximal oxygen uptake (VO₂max). Although laboratory-based graded exercise testing (GXT) with expired gas analysis remains the criterion method for VO₂max assessment, its practical application is limited by cost, equipment requirements, and the time needed to evaluate large groups. To overcome these limitations, the 20-meter multistage shuttle run test (20m-MST) has emerged as one of the most widely adopted field-based tools due to its simplicity and capacity for simultaneous group assessment. However, existing VO₂max prediction models based on the 20m-MST have largely been derived from small, sport-specific samples, limiting their generalizability to broader elite athletic populations. Therefore, the purpose of this study was to verify the validity of the 20m-MST and to develop a precise, field-applicable VO₂max prediction model for elite athletes across a wide range of ages, sexes, and sports disciplines. A total of 564 elite athletes (males = 336, females = 228; age range: 13-35 years) were recruited from 10 sports disciplines. Each participant completed both the 20m-MST and a laboratory-based maximal GXT with concurrent expired gas analysis, with a minimum interval of one week maintained between the two assessments. The GXT was conducted using the KISS Protocol, with VO₂max determined when at least two of five established physiological criteria were satisfied. Multiple regression analysis was conducted to develop a VO₂max prediction equation using shuttle run lap count, age, sex, and body weight as predictor variables, and both the intraclass correlation coefficient (ICC) and Bland-Altman analysis were employed to evaluate the reliability and limits of agreement between predicted and measured values. Correlation analysis revealed a statistically significant positive correlation between VO₂max and 20m-MST lap count across all subgroups (r = 0.700, p < 0.001). The regression model yielded a multiple correlation coefficient of r = 0.833, an explanatory power (R2) of 69.4%, and a standard error of estimate of 3.31 mL/kg/min. Validation demonstrated an ICC of r = 0.901, reflecting excellent agreement, with a mean difference of -0.031 ± 3.30 mL/kg/min and Bland-Altman limits of agreement of -0.17 ± 6.13% (95% CI: -0.30 to 0.24). The 20m-MST involves progressively increasing speed with repeated directional changes, indicating that both aerobic and anaerobic energy systems contribute to performance, and shuttle run results should therefore be interpreted as a composite indicator reflecting VO₂max alongside agility and anaerobic capacity. The inclusion of age, sex, and body weight as predictor variables allows for VO₂max estimation across both sexes and a broad age range within a single unified equation, representing a substantial improvement over previously reported models derived from limited samples. The developed prediction model demonstrated excellent validity and reliability, providing a practical and accurate tool for the assessment of cardiorespiratory endurance in elite athletes. Future research should consider refinements to the shuttle run protocol and the incorporation of correction factors for anaerobic energy contributions to further enhance the precision of VO₂max estimation in diverse athletic populations.