Rationale = Polyoxometalates (POMs) are remarkable oxo‐clusters forming compact highly charged anions. We measured their collision cross sections (CCS) in N2 with drift tube ion mobility spectrometry (DTIMS). These values were then used to calibrate a traveling wave ion mobility spectrometry (TWIMS) device and the accuracy of the calibration was tested. Methods = Six POM standards were analyzed by DTIM‐MS (Tofwerk, Thun, Switzerland) at different voltages to determine absolute DTCCS (N2) values. Five POM compounds (Lindqvist TBA2Mo6O19; decatungstate TBA4W10O32; Keggin TBA3PMo12O40; TBA3PW12O40 and Dawson TBA6P2W18O62) were used for the calibration of the TWIM‐MS instrument (Synapt G2 HDMS, Waters, Manchester, UK) and a sixth Dawson POM, TBA9P2Nb3W15O62, was used to compare the accuracy of the calibrations with POM or with polyalanine and dextran reference ions. Results = We determined 45 DTCCS (N2) values at 30°C or 60°C. Fourteen DTCCS (N2) values at 30°C were used to perform calibration of the TWIMS instrument. Better correlations were observed than when DTCCS values in helium from the literature were used. The accuracy tests on six ions of Dawson POM TBA9P2Nb3W15O62 led to relative errors below 3.1% while relative errors of 3.6% to 10.1% were observed when calibration was performed with polyalanine and dextran reference ions. Conclusions = Our novel calibration strategy for determination of CCS values of multiply negatively charged ions on TWIM‐MS devices based on DTCCS (N2) of standard POM structures covered a wider range of CCS and improved the accuracy to 2.1% relative error on average compared with 6.9% using polyalanine and dextran calibration.