Smartphone sensors have often been proposed as pervasive measurement systems to assess mobility in older adults due to their ease of use and low-cost. This study analyzes a smartphone-based application’s validity and reliability to quantify temporal variables during the single sit-to-stand test with institutionalized older adults. Forty older adults (20 women and 20 men; 78.9 ± 8.6 years) volunteered to participate in this study. All participants performed the single sit-to-stand test. Each sit-to-stand repetition was performed after an acoustic signal was emitted by the smartphone app. All data were acquired simultaneously with a smartphone and a digital video camera. The measured temporal variables were stand-up time and total time. The relative reliability and systematic bias inter-device were assessed using the intraclass correlation coefficient (ICC) and Bland-Altman plots. In contrast, absolute reliability was assessed using the standard error of measurement and coefficient of variation (CV). Inter-device concurrent validity was assessed through correlation analysis. The absolute percent error (APE) and the accuracy were also calculated. The results showed excellent reliability (ICC = 0.92–0.97; CV = 1.85–3.03) and very strong relationships inter-devices for the stand-up time (r = 0.94) and the total time (r = 0.98). The APE was lower than 6%, and the accuracy was higher than 94%. Based on our data, the findings suggest that the smartphone application is valid and reliable to collect the stand-up time and total time during the single sit-to-stand test with older adults.