Traditionally, GPs have been responsible for physical activity (PA) assessment within the general practice setting. Multiple questionnaires are available to support uptake of PA assessment but less than 30% of patients are assessed. A range of barriers hamper uptake. Evidence indicates that practice nurses (PNs) and patients are resourceful members of the general practice team but have been underutilised. This study assessed the validity and reliability of two instruments for assessing PA, administered by PNs and patients. The study aimed to identify robust tool(s) to support the evolving role of PNs and patients in prevention and management strategies in general practice. A purposive sample of PNs and patients from general practices in Sydney was invited to participate. The results of the PN- or patient-administered general practice physical activity questionnaire (GPPAQ) and the three-question physical activity questionnaire (3Q) were compared against accelerometer activity. The study examined agreement in classification of PA levels according to Australian PA recommendations. Validity showed low-moderate correlations between accelerometer and GPPAQ (rho≤0.26), 3Q (rho≤0.45). Seven-day test-retest reliability intraclass correlation coefficients (ICCs) were 0.82-0.95 for GGPAQ and 0.94-0.98 for 3Q. Agreement with PA recommendations was moderate for GPPAQ (kappa 0.73, 95% CI, 0.56-0.85) and fair for 3Q (kappa 0.62, 95% CI, 0.47-0.78). Although 3Q demonstrated higher correlation with accelerometry, GPPAQ demonstrated higher agreement with PA guidelines. Given GPPAQ showed reasonable rigour, it may prove useful for PN and patient use.