Volatile organic compounds (VOCs) affect secondary pollutant formation via active chemistry. Proton-transfer-reaction mass spectrometry (PTR-MS) is one of the most important techniques to study the highly variable spatial and temporal characteristics of VOCs. The response of protonated, adduct, and fragmented ions in PTR-MS in changing instrument settings and varying relative humidity (RH) requires rigorous characterization. Herein, we present dedicatedly designed laboratory experiments conducted to investigate the response of these ions for 21 VOCs, including 12 oxygenated VOCs and 2 nitriles, using the recently developed Vocus PTR-MS. Our results show that the focusing ion-molecule reactor (FIMR) axial voltage increases sensitivity by 3 to 4 orders of magnitude but does not significantly change the fractions of protonated ions. Reducing the FIMR pressure, however, substantially increases fragmentation. Applying a high radio frequency (RF) amplitude radially to the FIMR can enhance sensitivity by 1 to 2 orders of magnitude without affecting the protonated ion fractions. The change in big segmented quadrupole (BSQ) amplitude mainly affects sensitivity and protonated ion fraction by modifying ion transmission. The relationship between sensitivity and proton-transfer reaction rate constant is complicated by the influences from both ion transmission and protonated ion fraction. The protonated ions of most VOCs studied (19 out of 21) show less than 15% variations in sensitivity as RH increases from 1/45% to 1/485%, except for some long-chain aldehydes which show a positive RH variation of up to 30%. Our results suggest that the Vocus PTR-MS can reliably quantify the majority of VOCs under ambient conditions with varying RH. However, caution is advised for small oxygenates such as formaldehyde and methanol due to their low sensitivity and for long-chain aldehydes due to their slight RH dependence and fragmentation.